EXPANDED UTILITY OF RED-CELL DERIVED MICROPARTICLES (RMP) FOR TREATMENT OF BLEEDING

Abstract

Red blood cell membrane derived microparticles (RMP) are safe, economical, effective hemostatic agents in the treatment of a wide range of bleeding conditions and can, therefore, be considered as universal hemostatic agents. Effective RMP are produced from red blood cells using a high-pressure extrusion membrane shear process. The RMP can be lyophilized after production and retain activity even when stored at room temperature. RMP can be administered to original donors (autologous treatment), thus avoiding transfusion complications, or can be administered to blood type compatible recipients. RMP produced from type O, Rh negative red cells can be given to any person regardless of blood type. RMP can be administered to reduce excessive bleeding resulting from trauma, surgeries, invasive procedures and various bleeding disorders such as platelet disorders, either congenital or acquired, and coagulation disorders, either congenital or acquired. Administration of RMP prepared according to the invention demonstrates effectiveness in safely reducing bleeding.

Claims

1. A process for producing RMP comprising the steps of: suspending red blood cells in an aqueous diluent to form a cell suspension; pressurizing the cell suspension; extruding the pressurized cell suspension into a region of lower pressure to generate shear forces on the suspended red blood cells whereby the suspended cells are converted into a crude RMP suspension; and removing any whole red blood cells from the crude RMP suspension to make a final RMP suspension.

2. The process according to claim 1, wherein a French Press is used to pressurize and extrude the cell suspension.

3. The process according to claim 1, wherein whole red blood cells are removed from the RMP suspension by centrifugation.

4. The process according to claim 3, wherein RMP are removed from the crude RMP suspension by centrifugation.

5. The process according to claim 1, wherein whole red blood cells are removed from the RMP suspension by filtration.

6. The process according to claim 1 further comprising a step of washing the red blood cells with saline prior to the step of suspending.

7. The process according to claim 6, wherein the saline further comprises EDTA.

8. The process according to claim 1 further comprising a step of lyophilizing the final RMP suspension.

9. The process according to claim 1 wherein the red blood cells are from an autologous donor.

10. The process according to claim 1, wherein the red blood cells are selected from the group consisting of type ARh positive, type ARh negative, type BRh positive, type BRh negative, type ABRh positive, type ABRh negative, type ORh positive and type O, Rh negative.

11. The process according to claim 10, wherein the red blood cells are type ORh negative.

12. A process for treating and preventing excessive bleeding in a mammal caused by traumas, surgeries, invasive procedures or due to bleeding conditions including platelet or coagulation disorders, either congenital or acquired comprising the step of administering RMP to the mammal.

13. The process according to claim 12, wherein the platelet disorder is either thrombocytopenia or platelet dysfunction.

14. The process according to claim 13, wherein thrombocytopenia is caused by immune system, by drugs or agents, by chemotherapy, by systemic disease, or by bone marrow failure.

15. The process according to claim 13, wherein the platelet dysfunction is either congenital or acquired.

16. The process according to claim 15, wherein the platelet dysfunction is caused by a drug treatment.

17. The process according to claim 16, wherein the drug treatment is treatment with a drug that impairs platelet function.

18. The process according to claim 17, wherein the drug is selected from the group consisting of aspirin, clopidogrel, other antiplatelet drugs and other antiplatelet agents.

19. The process according to claim 12, wherein the coagulation disorder is congenital or acquired.

20. The process according to claim 19, wherein the coagulation disorder is caused an anticoagulant selected from the group consisting of Coumadin, heparin, inhibitors of prothrombinase complex and inhibitors of thrombin including dabigartran.

21. The process according to claim 20, wherein the low molecular weight heparin is enoxaparin or dalteparin.

22. The process according to claim 20, wherein the inhibitor of prothrombinase complex is fondaparinux or rivaroxaban.

23. A process for treating and preventing excessive bleeding in a mammal comprising the step of administering RMP in combination with other cell derived microparticles to the mammal.

24. The process according to claim 20, wherein the other cell derived microparticles are PMP.

Description

DESCRIPTION OF THE FIGURES

[0028] FIG. 1 is a graph showing thrombin generation by RMP as wells as by microparticles from other cells (note that the pattern of thrombin generation differs among RMP, PMP (platelet MP), EMP (endothelial MP) in terms of incubation time and amplitude of thrombin generation);

[0029] FIG. 2 is a bar graph demonstrating the relative absence of Tissue Factor (TF) expression on RMP as compared to microparticles derived from other cell types;

[0030] FIG. 3A is a bar graph demonstrating hemostatic effect of RMP produced using a high pressure extrusion device French Press (note that RMP shortened rat tail bleeding time and blood loss in rats with platelet dysfunction induced by the anti-platelet drug Plavix;

[0031] FIG. 3B is a bar graph demonstrating hemostatic effect of RMP produced using high-pressure extrusion devices from Avestin or Constant Systems (note that RMP produced by these two devices shortened rat bleeding time in a manner similar to those produced by French Press);

[0032] FIGS. 4A-H show TEG profiles of bleeding disorders in the presence or absence of RMP; FIG. 4A: Thrombocytopenia from aplastic anemia (platelet count 1,000/pL); FIG. 4B: Thrombocytopenia from ITP (platelet count 70,000/pL); FIG. 4C: Platelet dysfunction caused by aspirin; FIG. 4D: Platelet dysfunction caused by Plavix; FIG. 4E: Coagulopathy caused by Coumadin; FIG. 4F: Coagulopathy caused by Lovenox; FIG. 4G: Coagulopathy caused by Pradaxa; FIG. 4H: Congenital hemophilia A with a mild inhibitor;

[0033] FIG. 5 shows a TEG tracing with RMP and PMP alone and combination of RMP and PMP demonstrating shortening of R time, increasing A and enhancing MA indicative of a synergistic effect from the combination;

[0034] FIG. 6 is a graph showing that RMP increases procoagulance of factor VIII deficient plasma, corroborating the results shown in Table 2;

[0035] FIG. 7A is a graph showing that RMP enhance platelet aggregation at a low dose (0.2 pM) of ADP;

[0036] FIG. 7B is a graph showing that RMP enhance platelet aggregation at a low dose (0.3 mM) of arachidonic acid; and

[0037] FIG. 8 is a bar graph showing RMP enhanced platelet adhesion.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventors of carrying out their invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide methods for the production, storage and use of RMP.

[0039] Disclosed herein are unique hemostatic benefits of high-pressure shear produced RMP as effective and safe treatments for a wide variety of bleeding situations. Accordingly, in addition to treatment of bleeding resulting from thrombocytopenia, platelet dysfunction and surgical procedures and trauma, compositions and methods are provided for treating coagulopathy induced by anticoagulant drugs or systemic disease or clotting factor deficiency. These and other disorders that result in bleeding are treated by the administration of an effective amount of high-pressure shear RMP to a patient in need of treatment.

[0040] Effective dosages of RMP can be determined without undue experimentation by those of skill in the art and are generally expected to be between 10.sup.6 and 10.sup.12 particles/kg of body weight, more usually between 10.sup.8 and 10.sup.10 particles/kg. RMP may be administered in any suitable pharmaceutical composition according to the pharmaceutical arts, including normal saline or other physiologically acceptable buffers known to those of skill in the art, and optionally with additional therapeutic compounds, excipients and carriers as may be considered advantageous. The pH of the suspending buffer should generally be equal to or below 7.4.

[0041] RMP may be administered by any convenient and effective means known to those of skill in the art, particularly intravenously, or by direct application (e.g. topically, or by local injection) to a site where hemostasis is needed or desired. Such means will be known and/or easily determined without undue experimentation.

[0042] Further details about pharmaceutical compositions and administration of RMP can be found in the parent application (U.S. patent application Ser. No. 11/792,399) of the current application.

[0043] An improved method of RMP production is provided herein; this improved method employs shear induced by high pressure extrusion to enable economically feasible, large-scale RMP production without the use of additives and includes a method of long-term RMP storage. For RMP production by high shear principle, we used a French Press, but we also tested RMP produced by other instruments employing the same principle, such as those from Avestin and Constant Systems in vitro. These high pressure extrusion induced shear devices all generate RMP with similar procoagulant properties in vivo as shown in FIG. 3B. Table 1 compares the flow cytometric markers measured on RMP produced by the French Press with RMP produced by either a Constant System device or by an Avestin device. These results demonstrate that RMPs generated by different devices using similar high pressure extrusion are similar.

TABLE-US-00001 TABLE 1 Production RMP markers (Counts/uL) Device Annexin V Glycophorin A Ulex europaeus French Press 8,426 27,145 42,016 Constant Systems 7,547 22,271 33,286 Avestin 4,274 10,005 18,512

Example 1

Production of RMP (Laboratory Scale)

[0044] For a single batch, 30 mL packed cells from blood bank are diluted with 60 mL of isotonic saline containing 1.5 mM EDTA pH 7.4. Cells are washed 3 times with the same EDTA/saline by centrifuging 15 min. each at 750g at room temp. The final resuspension is brought to volume 60 mL, which is then drawn into the stainless-steel pressure cell of the French Press (Thermo-Electron Inc.) and then expelled at an internal pressure of 25,000 psi at rate 1.5 mL/min (achieved by adjustment of the needle valve). The resulting effluent was centrifuged at low speed (750g) to remove the small number of unbroken cells, and the supernatant was then centrifuged at 18,000g for 45 min. to sediment the RMP. One of ordinary skill in the art will appreciate that filtration and other separation techniques can be employed to remove the unbroken cells. The RMP were then washed at the same speed in isotonic saline (no EDTA) and refrigerated overnight prior to lyophilization. This procedure is >99% efficient in the sense that the starting red cells are almost entirely converted to uniform RMP of suitable size range.

Example 2

Potential for Scale-Up

[0045] This principle (shear induced by high-pressure extrusion) can be readily adapted to commercial-scale production. It was arrived at only after extensive experimentation with many other methods such as ionophore, sonic disruption, osmotic rupture, and others. The basic function of a French Press is to apply shear to fluid suspended cells. The cell suspension is placed in a pressure cell where pressure is applied by means of a hydraulic ram whereupon the pressurized suspension is forced (extruded) through the orifice of a needle valve into a region of atmospheric pressure. The great drop in pressure as the cells pass through the orifice applies great shear force to the biomembranes. The process is controlled by setting a target pressure on the hydraulic ram and controlling the needle valve to achieve a given flow rate. A drawback to the French Press is that the volume processed is limited by the size of the pressure cell (typically 100 mL) and the need to manipulate the needle valve accurately. Extensive study of French Press-treated membranes has demonstrated that the system generally produces inside out membrane vesicle. A number of related high-pressure shear systems obviate some of the disadvantages of the French Press. Pumped fluid homogenizers pressurize a larger volume of fluid and force it through a spring loaded valve into a region of atmospheric pressure. The effect is the same as the French Press except that the valve spring can be more reproducibly preset. Other pumped fluid homogenizers dispense with any type of valve and simply extrude the cell suspension (at a selected pressure) through a narrow channel or a fixed orifice into a region of atmospheric pressure. Both the pressure setting and the orifice diameter or channel diameter/length can be selected to achieve the desired results. Pumping in such systems can be achieved by reciprocating piston systems so that the device is essentially a self-filling French Press. Alternatively, the effluent stream from the high pressure extrusion system can be directed to impinge on a surface (e.g., a metal plate) to enhance the disruption of RBC. One of ordinary skill in the art can readily adjust the target pressure and the valve/channel openings of such devices to achieve production of high quality RMP.

Example 3

Lyophilization for Storage

[0046] An RMP batch (from Example 1) made the previous day was mixed with optimal amounts of albumin, glucose, sorbitol and/or trehalose, then pipetted in 1.0 mL aliquots into 2 mL lyophilizing vials, then placed in the tray of a Triad Freeze Dry System (Labconco, Inc.). The instrument operation is divided into programmable segments. Our experiments established the following optimal program settings. Samples are first subjected to a 4-hour Pre-freeze phase, with brief vacuum, during which temperature drops to 75 C. Segment 1. After switching to full vacuum (<0.07 mbars), temperature is ramped up (at 0.27 C./min) to 30 C. and held for 2-hr. Segment 2. Temperature is slowly increased (at 0.04 C./min) to 25 C., and held for 2-hr. Segment 3. Temperature is ramped to 15 C. at 0.04 C./min, and held for 12-hr. Final Step. Vials are sealed in vacuum; rubber stoppers were pre-placed on the vials and pressed on to seal by pressure plate. After removing vials, aluminum tab seals are crimped on; vials are stored at room temp. Activity recovered is equal or superior to freezing at 70 C. for at least 90 days, with no decline observed. Of course, the material can be stored at low temperature to ensure long-term activity, but our tests indicate that even a protracted period of storage at room temperature does not destroy activity.

[0047] The inventors found that RMP lyophilized in this manner show excellent efficacy in vitro and in animal models, as compared with fresh RMP (prior to freezing or lyophilizing). Persons of ordinary skill in the art will appreciate that some variation in the procedural parameters will produce an equally superior product and that further improvement in properties of the RMP product may be made by routine adjustments of the procedural parameters. As mentioned above, the lyophilized RMP show no decline of activity for at least 90 days at room temperature.

Example 4

Thrombin Generation Assay (TGA) (FIG. 1)

[0048] This assay was based on the method and software of Hemker et al. [6], termed Calibrated Automated Thrombin (CAT) generation assay [7], adapted by the inventors to measure activity of microparticles [8]. The method measures a change in signal when a fluorescent substrate for thrombin is cleaved. Procedure: In the experiment shown, RMP were generated by exposure of washed fresh RBC from normal donor to calcium ionophore A23187 (10 M) for 1 hr. In a later study (not shown), we generated RMP by means of the high pressure shear extrusion method described above and confirmed that RMP produced using both methods show similar hemostatic activity. PMP (platelet microparticles) were generated by exposure of human platelet rich plasma to ADP (10 M) for 30 min. EMP (endothelial microparticles) were obtained from the supernatant of cultured human renal microvascular endothelial cells activated by tumor necrosis factor (TNF-) (10 nM) for 24 hrs. In all cases unbroken cells were removed by centrifugation (700g for 15 min), and the resulting microparticles were sedimented (15,000g for 30 min) and pellets were washed twice, then resuspended to equal microparticle concentrations (110.sup.8 counts/mL, final concentration) based on counts by flow cytometry. Then 10 L of microparticles were mixed with 90 L of corn trypsin inhibitor (25 g/mL)-treated particle free plasma (PFP) after which the CAT test was initiated by addition of calcium. Relevant data are chiefly the lag time, seen on the x-axis, and peak thrombin generated, seen on the y-axis in units/nM. The inventors' laboratory was first to document thrombin generation by RMP [5]. The time-course of thrombin generation by RMP is distinctive, as shown. Note the long lag-time of RMP compared to PMP and EMP, which is attributed to the absence of tissue factor (TF). This is a great advantage of RMP as hemostatic agent. RMP, however, generate equally strong amplitude once coagulation is initiated. As seen in FIG. 1, the mode of thrombin generation differed among EMP, PMP and RMP. Accordingly, combined use of EMP and PMP along with RMP could be of benefit in certain clinical settings. MP types with distinct hemostatic property can be combined to exert optimal hemostatic efficacy in reducing bleeding. This is an important rational for supporting MP combination therapy.

Example 5

Absence of TF Expression on RMP (FIG. 2)

[0049] The method used was flow cytometric detection of TF antigen using PE/Cy5-labeled anti-TF (American Diagnostica). PMP, EMP and RMP were prepared as described in the previous section. LMP (leukocyte microparticles) were prepared by incubating neutrophils (510.sup.6/mL) with lipopolysaccharide (LPS, 10 ng/mL final concentration). The neutrophils were isolated by standard procedures (Ficol-Hypaque density centrifugation). All microparticles were adjusted to equal concentration (110.sup.8/mL). TF was measured in flow cytometry by double-staining (anti-TF and FITC-Ulex lectin) prior to aspiration into the flow cytometer. Event detection was triggered by the green fluorescent signal. Results are given as percentage of microparticle-positive events that were also positive for TF. It is apparent that TF expression on microparticles would promote systemic thrombosis. However, TF can benefit local hemostasis at the site of injury. Accordingly proper combination of RMP and tissue factor expressing microparticles will have therapeutic advantage of certain bleeding condition as described above.

[0050] The figure demonstrates that TF is not detected on RMP but is easily detected on other cell-derived microparticles: those from platelet (PMP), endothelia (EMP) and leukocytes (LMP). This result is consistent with literature identifying, TF on all microparticles except t RMP. TF is the main physiological initiator of coagulation and thrombosis. So that the absence of TF on RMP indicates a high degree of safety, i.e., inability to generate thrombin, except a local site of injury where TF is present. In other words, RMP are not efficient in initiating thrombin generation, but contribute robustly to its generation upon exposure of TF, such as at a bleeding site, thus providing effective hemostasis locally but not systemically.

[0051] Because RMP, PMP, EMP, LMP have different hemostatic property as demonstrated in FIGS. 1 and 2, use of RMP combined with other MP such as PMP can exhibit a synergistic effect. FIG. 5 demonstrates that combination of RMP with PMP exhibit synergistic effect in hemostasis when examined by TEG. Use of the combination shortened clotting time (R) and increased speed of clot formation (A) and enhanced clot strength (MA) compared to RMP or PMP alone.

Example 6

Use of RMP to Treat Platelet Dysfunction (FIG. 3)

[0052] In the parent of the instant application, applicants presented data showing that infusion of RMP shortened tail bleeding time in thrombocytopenic rats.

[0053] The question addressed by this study was whether RMP were able to reduce bleeding from platelet dysfunction as well. We induced platelet dysfunction in rats by intravenous injection of Plavix (10 mg/kg). FIG. 3 shows that platelet dysfunction induced by Plavix increased bleeding time and blood loss by at least 5-fold compared to controls, and some animals did not stop bleeding at all and expired. However, all eight animals treated with RMP (dose of 2.010.sup.9/kg) showed almost complete normalization of bleeding time and blood loss.

[0054] The experiments were conducted in Sprague-Dawley rats. The procedure was begun by weighing the animal (range 250-350 g) and anesthetizing them by adding 1-2 mL of Isoflurane to a sealed container in which the rat was placed. Once sedated, the rat was placed on a special platform that allows a technician to intubate it. The animal constantly received oxygen and Isoflurane from a respirator to maintain anesthesia. The animal was then affixed to a surgical board and a rectal thermometer is used to monitor body temperature. A heating pad was placed under the board to maintain body temperature. When stable, the neck of the animal was shaved and a small incision was made to find the jugular vein and the carotid artery. Any slight bleeding during this procedure was stopped with a cautery tool. Both the jugular vein and the carotid artery were cleaned and isolated from surrounding connective tissue. Before inserting the cannulas, blood flow was controlled by tying a section of the vein and artery. Once the cannulas were in place, they were secured with sutures and the incision closed. The jugular vein was later used to inject test substances (RMP, medications) and the carotid artery was used to supply vital fluids (lactated Ringer's) and to monitor heart rate and blood pressure. This allows the rat to survive for the required 3-4 hours. Bleeding was initiated by cutting a 2 mm segment of a toe and all blood was collected in tube for total blood loss and time to cease bleeding was measured.

[0055] Baseline data was obtained for n=23 untreated animals (controls; at left). Platelet dysfunction was induced by infusion of clopidogrel (Plavix) at a dose of 10 mg/kg through the catheter 10 min. prior to bleeding. The center bar shows increased bleeding time and blood loss due to clopidogrel. The error bar is +/SEM. The left panel is a comparison of bleeding time between controls, animals treated with Plavix, and animals treated with both Plavix and RMP. The right panel is a comparison of total blood loss between the same groups. RMP administered at a dose of 2.010.sup.8/kg in volume 400 L five minutes prior to bleeding gave correction of bleeding time and blood loss to near-normal range, as shown (right bars). Note the near normalization of bleeding time and blood loss by RMP infusion.

[0056] In our animal model, improved instrumentation was used to measure heart rate, blood pressure, respiration and temperature. Vital signs were closely monitored before and after RMP infusion. Bleeding time and total blood loss were measured after cutting toe, as described. All treated animals were observed for up to four hours post-treatment (under anesthetic). None of the RMP-treated animals showed indication of thrombosis or signs of adverse change in temperature, heart rate, respiration, or neurological signs. This included two cases tested at double the full effective dose; no further reduction in bleeding time (below normal) was observed. This indicates absence of prothrombotic effects, and suggests complete safety in all cases studied thus far. Since RMP do not carry tissue factor (TF), there is no theoretical reason to expect adverse effects. The apparent absence of adverse effects even at high dose is considered to be an important advantage of RMP compared to other products intended for this purpose.

[0057] We also studied the effectiveness of RMP generated by different high pressure instruments (e.g., Avestin, Constant system) using the rat model described above. These RMP were as effective in shortening tail bleeding time as were RMP produced by the French Press high pressure instrument (as shown in FIG. 3A). Table 1 compares the flow cytometric markers measured on RMP produced by the French Press with RMP produced by either a Constant Systems device or by an Avestin device. These results demonstrate that RMP produced using various high pressure extrusion devices are similar.

Example 7

Clinical Study of RMP on Various Bleeding Disorders by Means of TEG

[0058] Bleeding time (BT) was formerly used to predict risk of bleeding at surgery but is now replaced by thromboelastograph (TEG) measurements because of superior reproducibility and the additional important data provided by TEG instruments [9-11]. TEG is the best in vitro assay to predict risk of bleeding in vivo and its value in assessing hemostasis is well accepted. TEG measurement is increasingly utilized to assess bleeding with invasive procedures or surgery such as renal biopsy [8], cardiovascular surgery [9, 10] and trauma [11]. It is such a widely accepted practice now for surgeons to pre-order blood for transfusion based on TEG abnormality in an effort to prevent excessive bleeding.

[0059] TEG Protocol:

[0060] Blood samples were drawn in citrated vacutainers from patients with various bleeding disorders. Blood samples were pipetted (330 L) into the cup of the TEG Haemoscope (Haemonetics Co.), then 10 L of saline (control, without RMP). RMP produced through high-pressure extrusion was added to each well at the dose of 6.810.sup.7 particles to bring the final RMP concentration to 210.sup.8/mL. The reaction was initiated by adding 10 L of CaCl.sub.2. TEG tracings were obtained without and with RMP, and alteration of TEG parameters by addition of RMP was evaluated. The quantity of RMP added was calculated from the therapeutic dose determined in animal experiments.

[0061] FIGS. 4 A-G are examples of clinical studies to evaluate hemostatic property of RMP. It should be noted that TEG tracings without RMP are shown in unbolded solid lines and tracing after addition of RMP are shown in bolded broken lines. In each figure, changes in R, A and MA are compared in the boxes next to figures. These findings indicate potential value of RMP to treat various bleeding disorders. The applicants found that TEG gave results which corresponded closely to in vivo (animal) studies, in particular, results with Plavix, building confidence that TEG is a good surrogate for in vivo studies. This confirms literature that TEG correlates closely with bleeding time in vivo (human). TEG is also more reproducible, faster, and supplies additional important measurement parameters [9-11].

[0062] Parameters Measured.

[0063] TEG measures the delay of initiation of clotting (R), the rate of clot formation (a, angular rise rate), and maximum amplitude (MA). A high risk of bleeding is detected as a high R with a low a, or low MA measure value.

[0064] In aplastic anemia (FIG. 4A), bone marrow fails to produce sufficient red cells, white cells and platelets; this patient had fewer than 1% of normal platelets (1,000/L vs. normal value of 250,000/L). The figure shows almost no clotting in absence of RMP, but with a therapeutic dose of RMP a nearly normal trace is seen as indicated by fast initiation (R=9.7 min, from 53.5), a rate of rise (a=52.8, from 1.3) and an increase of MA (from 2.2 to 45.5) as detailed in the figures' side boxes.

[0065] In ITP (Idiopathic Thrombocytopenic Purpura) (FIG. 4B), platelets are destroyed by an autoimmune reaction; this patient had only 28% of the normal number of platelets. In absence of RMP, clotting is delayed nearly three-fold longer than normal (R=23.9) and rate is weak as seen in the low slope (a=19.5). A normal profile is restored by therapeutic dose of RMP (R=8.3, a=61.8).

[0066] In this patient (FIG. 4C), platelet dysfunction was due to aspirin, which inhibits platelet function and can result in serious bleeding, especially in combination with other disorders or medications. Note the elimination of the prolonged lag time (R) by RMP.

[0067] In this case (FIG. 4D) platelet dysfunction was due to therapy with Plavix (clopidogrel), which inhibits platelet function as does aspirin but by a different mechanism (blockade of ADP receptors). Despite a different mechanism of platelet dysfunction, RMP corrected the prolonged lag time.

[0068] Coumadin (a.k.a. Warfarin) is most widely used blood thinners prescribed for prevention and treatment of thrombosis. It acts by preventing effective production of the several clotting factors that require vitamin K. Overdose of Coumadin, which happens commonly, can lead to serious bleeding. In this patient (FIG. 4E), notice that RMP fully corrected the prolonged lag time (R) and slow rate (a). Platelet counts were normal in this patient.

[0069] Many new anticoagulants will soon come to market. The old as well as the new anticoagulants belong to one of four groups: 1) Vitamin K antagonists such as Coumadin; 2) Heparin and low molecular weight heparin; 3) inhibitors of thrombin (anti-factorlla) such as dabigatran; and 4) inhibitors of prothrombinase complex (anti-factor Xa) such as fondaparinux and rivaroxaban. The new anticoagulants are increasingly employed. Low molecular Heparin (LMWH) and especially enoxaparin (Lovenox) and dalteparin (Fragmin) are now widely used. These new anticoagulants do not have antidotes, therefore hemorrhagic complications from new anticoagulants is very problematic. FIG. 4F shows that RMP correct coagulation abnormality induced by Lovenox. Similar correction by RMP was seen in other LMWH such as dalteparin (Fragmin) and fondaparinux (Arixtra) as well as dabigatran (Pradaxa), a new oral thrombin inhibitor (FIG. 4 G) which is more convenient and practical to use than existing anticoagulants. RMP corrects clotting abnormalities induced by anticoagulants from any of the four groups. Therefore, RMP should be effective against bleeding resulting from any new drugs belonging to one of these groups.

[0070] Hemophilia A is an inherited disorder marked by ready bleeding due to low levels of functional factor VIII (FVIII). Painful and debilitating bleeding in the joints and mucous membranes are common, and brain hemorrhage can be fatal. Treatment is infusion of FVIII concentrates but this treatment is often ineffective due to formation of inhibitors of the FVIII administered. This patient (FIG. 4H) had congenital Hemophilia A and developed mild inhibitor. The figure shows that RMP corrected prolonged lag (R) and normalized rate (a) of clotting.

Examples 8

Mode of Action of RMP in Hemostasis

[0071] To investigate how RMP exert diverse actions in hemostasis, we mixed RMP with factor deficient plasma and studied alteration of procoagulance with TEG. Addition of RMP to factor deficient plasma shortened clotting time (R), increased rated of clotting (a) and enhanced clot strength (MA). However degree of increasing procoagulance by RMP differed between clotting factor deficiency. Effects of RMP in Factor II, V, VII, VIII, IX, X, XI XII, XIII deficiency are shown in the Table 2 [12]. The increase in procoagulance by RMP was most pronounced in Factor VIII (see FIG. 6) and Factor IX deficient plasmas where RMP could correct deficiencies as low as 5-10% of the normal levels of the clotting factors. In all cases addition of RMP resulted in a significant correction of clotting time.

[0072] Other Bleeding Conditions Seen Corrected.

[0073] RMP was observed to correct the abnormality in the following cases judged by TEG: (i) Patients with mild inhibitors to Factor IX, XI; (ii) patients treated with heparin, low molecular weight heparin (Lovenox, Fragmin) and Arixtra; (iii) patients with DIC; (iv) chronic liver disease; (v) thrombocytopenia from bone marrow failure such as aplastic anemia, myelodysplastic syndrome, leukemias. (data not shown).

[0074] FIGS. 7A and 7B show the effect of RMP on platelet aggregation and adhesion. At low concentrations of ADP (FIG. 7A) and arachidonic acid (FIG. 7B), RMP enhanced platelet aggregation measured by Chrono-Log aggregometry. We also studied the effect of RMP on platelet adhesion by a cone-well device (Diamed IMPACT-R), where RMP enhanced platelet adhesion by increasing the sizes of aggregates. FIG. 8 shows the enhancement in platelet adhesion. Note the statistically significant (* P=0.02) increase in size of aggregates of platelets by RMP at shear rate (S.sup.1)=1800, that resemble venous blood flow in vivo [12].

TABLE-US-00002 TABLE 2 RMP enhance procoagulance in factor deficient plasmas. R(min) A (degree) MA (mm) F II RMP 8.7 63.6 33 RMP+ 8.4 68.1 37 0.3 4.5 4 F V RMP 11.2 62.1 29.2 RMP+ 11.1 64.8 28.7 0.1 2.7 0.5 F VII RMP 11.6 57.7 29.6 RMP+ 11.3 62 25.5 0.3 4.3 4.1 F VIII RMP 30.8 23.1 26.3 RMP+ 16.6 52.6 29.7 14.2 29.5 3.4 F IX RMP 77.2 1.1 7.2 RMP+ 28.2 12.6 27.8 49 11.5 20.6 F X RMP 11.2 62.1 29.1 RMP+ 11.1 64.8 28.7 0.1 2.7 0.4 F XI RMP 14.8 55.6 27.6 RMP+ 12.2 50.2 47.7 2.6 5.4 20.1 F XII RMP 13.1 32.9 25.4 RMP+ 11.1 55.3 35.2 2 22.4 9.8 F XIII RMP 8.5 56.2 20.8 RMP+ 8.2 57.7 24.1 0.3 1.5 3.3

[0075] The following claims are thus to be understood to include what is specifically illustrated and described above, what is conceptually equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the invention. Those skilled in the art will appreciate that various adaptations and modifications of the just-described preferred embodiment can be configured without departing from the scope of the invention. The illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.

REFERENCES

[0076] References and publications cited herein are hereby incorporated by reference [0077] 1. Hannon T, Gjerde K P: The contemporary economics of transfusions. In Perioperative Transfusion Medicine; Speiss R D, Spence R K, Shander A (eds.), Lippincott Williams and Wilkins, p. 13 (2006). [0078] 2. Mannucci P M: Desmopressin (DDAVP) in the treatment of bleeding disorders: the first 20 years. Blood 1997; 90(7): 2515-21, [0079] 3. Hedner U: NovoSeven as a universal hemostatic agent. Blood Coagul Fribrinolysis 2000; 11(Suppl 1): S107-11. [0080] 4. Hawskworth J S, et al: Evaluation of lyophilized platelets as an infusible hemostatic agent in experimental non non-compressible hemorrhage in swine. J Thromb Haemost 2009; 7(10): 1663-71. [0081] 5. Jy W, Bidot L, Jimenez J J, Horstman L L, Bang J, Lin A, ZambranoJr W, Ahn E, Ahn Y-S: Thrombin generation profiles are qualitatively and quantitatively distinct in microparticles derived from red cells (RMP), platelets (PMP), and endothelia (EMP). Blood 2006; 108(11): 499a (Ab1759). [0082] 6. Hemker, H. C.: Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 33 (1), 4-15 (2003). [0083] 7. Luddington, R. and Baglin, T.: Clinical measurement of thrombin generation by calibrated automated thrombography requires contact factor inhibition. J Thromb Haemost 2 (11), 1954-1959 (2004). [0084] 8. Bidot, L., Jy, W., Bidot Jr, C., Jimenez, J. J., Fontana, V., Horstman, L. L and Ahn, Y. S.: Microparticle-mediated thrombin generation assay: increased activity in patients with recurrent thrombosis. J Thromb Haemost 6, 913-919 (2007). [0085] 9. Davis C L, Chancier W L: Thromboelastography for the prediction of bleeding after transplant renal biopsy. J Am Soc Neph 1995; 6(4): 1250-5. [0086] 10. Ronald A, Dunning J: Can the use of thromboelastography predict and decrease bleeding and blood and blood product requirements in adult patients undergoing cardiac surgery? Interact CardioVas Thorac Surg 2005; 4:456-63. [0087] 11. Plotkin A J, Wade C E, Jinkins D H, et al: A reduction in clot formation rate and strength assessed by thromboelastography is indicative of transfusion requirements in patients with penetrating injuries. J Trauma 2008; 64(2 Suppl.): S 64-8. [0088] 12. Ahn Y S, Jy W, Johansen M, Horstman L L: Red cell derived microparticles (RMP) as hemostatic agent to treat bleeding disorders: The mode of action of RMP. JTH (Suppl. 2): 269(2011) (Presented at the International Society of Thrombosis & Hemostasis, Jul. 26, 2011, Kyoto, Japan).