Patent Abstract:
compositions comprising membrane - derived and synthetic microparticles that induce platelet aggregation and are useful for treating bleeding disorders , particularly those involving platelet dysfunction . microparticles include endothelial derived microparticles , platelet derived microparticles , erythrocyte derived microparticles , and synthetic microparticles , and are used to treat disorders such as thrombocytopenia caused by chemotherapy .

Detailed Description:
we have previously shown that vwf is bound to subspecies of emp ( 32 ), but the functional significance was not known . the results described hereinbelow demonstrate that platelet - emp interaction is mediated through vwf , and compare the stability of the platelet aggregates formed by emp to those formed by normal plasma , humate - p , and ttp plasma . in addition , we studied the effect of vwf - positive emp on the ristocetin - induced aggregability of plasma from patients with von willebrand disease ( vwd ). further , we have compared and correlated the size of the vwf multimers between emp - bound vwf vs . soluble vwf from normal and ttp plasma , and humate - p with the stability of the platelet aggregates . additionally , we present results from two patients with severe itp correlating high levels of rmp with a reduced number of bleeding episodes compared to the average population of itp patients , and in vitro laboratory data demonstrating that rmp are effective hemostatic agents . human cultured endothelial cells of renal or brain , and coronary artery origin were obtained from cell systems ( kirkland , wash .). fitc - labeled anti - cd62e ( clone 1 . 2b6 , cat . # f - 0674 ) was obtained from sigma ( st . louis , mo .). anti - cd42b ( clone sz2 , cat . # im0409 ) and cd41 ( cat . # im0649 ) were obtained from beckman - coulter ( miami , fla .). hrp - conjugated anti - vwf ( cat # ahp 062 - p ) was purchased from serotec inc . ( raleigh , n . c .). gel electrophoresis reagents and instruments were obtained from bio - rad ( richmond , calif .). ristocetin ( sigma , st . louis , mo .) was purchased from chrono - log . humate - p ®, a therapeutic reagent containing concentrated factor viii and vwf multimers , was obtained from aventis - behring ( marburg , germany ). other chemicals were purchased from sigma ( st . louis , mo .). preparation of emp and platelets human cultured endothelial cells ( ec ) of renal , brain , or coronary origin were activated with tnf - α ( 10 ng / ml ) for 24 hr to induce emp generation ( 13 ). the culture supernatants were then centrifuged at 15 , 000 × g for 30 min to sediment emp , which were then washed 3 × with pbs buffer and re - suspended in pbs to 1 / 10 original volume . concentration of emp was measured by flow cytometry using fitc - labeled anti - cd62e as described by jimenez et al . ( 33 ). washed platelets were prepared by centrifuging platelet - rich plasma ( prp ) at 600 g for 10 min in the presence of 10 mm of egta and 1 μm of pge 1 . the pellets were washed twice with pbs , and then suspended in pbs at 1 × 10 8 / ml . assay of emp - platelet interaction and ristocetin - dependent aggregation by flow cytometry . emp at 5 - 100 × 10 6 / ml final concentration were incubated with normal washed platelets at 1 × 10 7 / ml final , in the presence or absence of ristocetin ( 1 mg / ml ) for 10 min with gentle orbital shaking ( 100 rpm ). binding of emp to platelets was assessed by co - expression of emp marker cd62e with platelet marker cd41 in flow cytometry . in those experiments , plasma was used from 1 % to 15 % as indicated ; and humate - p from 0 . 02 to 0 . 4 u / ml as indicated . platelet aggregation was measured by flow cytometry ( 34 ) by counting the number of free platelets (& lt ; 5 um ) shifted to a bit - map representing platelet aggregates (& gt ; 5 um ). the flow rate of the coulter xl flow cytometer was at medium setting and discriminator was forward scatter ( fs ), level 3 . the number of platelets was calibrated with standard beads with known concentrations . a decrease in singular free platelets ( accompanied by increase in number of platelet aggregates ) was observed when ristocetin was present in the plasma . at maximal plasma or humate - p plus ristocetin ( 1 mg / ml ), only & lt ; 5 % of platelets remain singular ( free ). reduction of number of singular platelets with and without ristocetin was measured as an indicator of platelet aggregate formation rather than counting the number of micro - aggregates because the latter is ambiguous owing to heterogeneous size distribution and sticking to the flow chamber and tubing . dissociation of ristocetin - induced platelet aggregates after 20 minutes of platelet aggregate formation at room temperature , the samples were diluted with pbs ( 1 : 20 volume ratio ) to induce a time - dependent dissociation . increases of free platelet counts after dilution were monitored at intervals to determine the time - course of dissociation of platelet aggregates by flow cytometry . vwf multimer analysis . the method of raines , et al . ( 36 ) was employed with minor modifications as follows . cooling during electrophoresis was accomplished by resting the horizontal gel electrophoresis apparatus on an aluminum block immersed in ice - water slurry and the buffer chambers were also on ice . several agarose gel concentrations were tested and 0 . 8 % was found to be optimal for showing a wide range of multimer sizes . western blotting was according to raines et al except that the anti - vwf was pre - conjugated with hrp ( serotec inc ., raleigh , n . c . ; cat # ahp 062 - p ) and was used at 1 : 500 dilution ( 50 ul in 25 ml ). the proteins in the gel were transferred into pvdf membrane by capillary diffusion with layers of paper towels on top of pvdf membrane overnight with pbs as transfer buffer . the pvdf membrane was then blocked with 0 . 5 % casein solution . staining of the pvdf membrane was accomplished by the method of nakane ( 37 ) using dye 4 - chloro - 1 - napthol ( 4cn ; sigma , cat . # c - 8890 ) prepared fresh by dissolving 30 mg in 5 ml of ethanol , then bringing to volume 100 ml by adding 50 mm tris buffer ph 7 . 6 containing 0 . 03 % h 2 o 2 ( 1 ml of 3 % h 2 o 2 in 100 ml ). clinical studies . for examples 4 - 6 , citrated blood was obtained from four ttp and four type i vwd patients . the four ttp patients all presented with the classic triad of ttp ; severe thrombocytopenia ( platelet count & lt ; 2 × 10 7 / ml ), microangiopathic hemolytic anemia , and mental dysfunction . the type i vwd patients were characterized by low vwf total antigen and deficient ristocetin cofactor activity . the protocol was approved by the institutional review board , and informed consent was obtained from the patients . for example 8 , blood ( citrated to optimize preservation ) was obtained from atypical (“ nonbleeding ”) itp patients , characterized in standard clinical studies , and compared to normal controls and typical itp patients . statistical analysis . for comparing three or more groups , one way anova was use to determine the p values . if p & lt ; 0 . 05 , then two tailed student &# 39 ; s t test was used to analyze the significance ( p & lt ; 0 . 05 ) of difference between the means of two groups . in cases where the data failed the normality test , then the mann - whitney rank sum test was used . all data analyses were performed using windows - based program , statmost . platelet microparticles may be prepared by at least three different methods , as indicated below , and as are well known to one of skill in the art . 1 ) blood - bank source . stored platelets release abundant pmp with time ( see section 4 . 5 , “ platelet storage lesion ,” of reference 1 ) this method may be utilized to make use of platelet concentrates that have been expired by up to 5 days , which are normally discarded . procedure : platelets expired by 1 - 5 days are sedimented by centrifugation 10 min at 200 × g , room temperature , sterile . observing sterile technique , the supernatant is diluted to double volume by adding pbs / citrate , then the microparticles are sedimented by centrifuging 8 , 000 × g for 30 min . the resuspended pmp may be refrigerated for storage , then re - washed prior to use . resuspend in desired i . v . medium ( e . g . saline ). 2 ) ultrasonic method . pmp may be obtained in high yield by sonic disruption . procedure : fresh ( or recently expired ) platelets at physiologic concentration ( 2 . 5 × 10 5 / ul ), previously washed 2 × in pbs buffer with 1 mm edta / 5 mm mgso 4 ( to minimize aggregation ), are exposed to ultrasonic disruption [ branson instruments ; 5 mm titanium probe ] in a 50 ml polypropylene tube for 3 - 5 bursts of 3 - 5 seconds each at room temperature . residual platelets and debris are removed by centrifugation and the pmp in the supernatant are sedimented by high - speed spin , then washed as above . sterile technique adequate for animal studies consists of a cotton plug in a hole drilled in the cap of the tube sufficient to admit the sonic probe , which is wiped with alcohol swab prior to inserting through the sterile cotton collar . for human use , appropriate sterile techniques are known to those of skill in the art . 3 ) calcium activation method . in normal physiology , a rise in cytosolic calcium is the final common step of all pathways leading to cell activation . use of reagent calcium ionophore , such as a23187 , creates a pore in the membrane which selectively admits calcium from the external medium , causing abundant pmp release . the resulting may be more physiologically relevant . procedure . the method of arnout et al ( 42 ) can be used to prepare pmp , and the resulting pmp express significant vwf by electrophoresis / blotting [ unpublished ]. briefly , 1 . 0 ml washed platelets are suspended at 2 . 5 × 10 5 / ul in hepes / saline ph 7 . 4 , then ionophore a23187 sufficient to make 1 umol / l final is added ( prediluted from alcohol stock solution ) in 50 ul buffer , sufficient calcium to make 2 mm final , then gently agitate for twenty minutes at room temperature . centrifuge to remove heavy debris , and recover pmp from supernatant as above . alternate : platelets may be permeabilized with saponin instead of ionophore if there is any concern that ionophore will persist in the pmp ; then proceed as above . alternate : platelets may also be activated with thrombin + collagen , reported to have potency similar to ionophore activation . as described in i ( 1 ) above , patients &# 39 ; own blood or blood - bank blood is also known to naturally shed abundant rmp . therefore , they may be harvested exactly as described in i ( 1 ). fresh rbc as described in i ( 2 - 4 ) as well as stored rbc can be used for generation of rmp . liposome approach ( synthetic mp ). on the assumption that vwf in conjunction with procoagulant phospholipids ( pl ) are by themselves sufficient to afford significant hemostatic protection in thrombocytopenic patients , vwf - cephalin ( or lecithin ) liposomes should be efficaceous . the experience of the inventors shows that both soybean and egg lecithin are almost as active in coagulation assays ( tissue factor , also lupus anticoagulant ) as is cephalin , the crude brain lipid traditionally used for this purpose . the substantial advantage of this approach is limited immunogenic proteins , other than vwf itself . procedure : briefly , vwf concentrate ( such as humate p ® or equivalent prepared in - house from plasma ) is exposed to ultrasonic energy in the presence of liposomes of cephalin or lecithin , in concentrations well known in the art . under appropriate conditions , essentially all of the vwf becomes tightly associated with the liposomes , either on the surface or transmembranally . optimum conditions can be determined by those of skill in the art without undue experimentation . platelet aggregation induced by emp . emp were incubated with normal washed platelets as described above . the presence of emp at 4 × 10 7 / ml final induced strong platelet aggregation which was dependent on ristocetin . as seen in fig1 , the degree of ristocetin - dependent platelet aggregation caused by emp was similar to that caused by 8 % normal plasma . in the absence of ristocetin , negligible platelet aggregates were formed with either emp , humate - p , or normal plasma . both emp - induced and plasma - induced platelet aggregation was inhibited by anti - cd42b blocking mab . these results demonstrate that emp induced platelet aggregate formation that is vwf - dependent . the dose - response curves of platelet aggregation induced by emp , normal plasma , or humate - p are shown in fig2 . it is noted that the shapes of the curves for all three agents are similar . since both the platelets and the emp were pre - washed and essentially plasma - free , these results demonstrate that emp - bound vwf can substitute for soluble vwf in plasma or humate - p in inducing full platelet aggregation with ristocetin . the data indicate that 50 % aggregation occurs with 3 . 5 % plasma , equivalent to 1 × 10 7 / ml of emp , and to 0 . 075 u / ml of humate - p . to further confirm the existence of microparticle - bound vwf , we tested the effect of filtration through 0 . 1 μm filter , which is known to retain the majority of emp . as shown in fig3 , this filtration largely abolished emp - induced platelet aggregation but had no significant effect on normal plasma - or humate - p - induced platelet aggregation . emp from endothelia of different origins . we compared aggregation activity of emp from three different sources of ec : microvascular renal and brain , and macrovascular coronary artery . all three ec were cultured under conditions similar to those detailed previously ( 13 , 33 , 34 ) and were stimulated with the same concentration of tnf - α for 24 hr . emp were collected and washed as described above , counted by flow cytometry , and adjusted to equal concentrations . table 1 shows the relative specific activities of the emp from these three sources in inducing vwf - dependent platelet aggregation with ristocetin . it is seen that emp derived from renal or brain microvascular ec were more potent than those from coronary artery ec . this is consistent with our previous findings that renal or brain emp contained higher percentage of vwf + emp ( 33 ) and with the fact that the clinical manifestations of abnormally active vwf are mainly related to microangiopathic thrombosis . emp obtained from renal , brain and coronary artery endothelial cells ( ec ) as described in the “ methods ” section were adjusted to equal concentrations prior to evaluating their proaggregatory activity in the presence of ristocetin . the table shows that emp from different ec lines exhibited different activities in ristocetin - induced platelet aggregation , in the following order , renal & gt ; brain & gt ;& gt ; coronary ec . n = 4 , mean ± s . d . * indicates p & lt ; 0 . 01 as the “ renal emp ” or “ brain emp ” group compared to the “ coronary emp ” group . assessment of aggregate stability . in the course of pilot studies , we observed that when platelet aggregates induced by plasma plus ristocetin were diluted 20 - fold with pbs buffer , the aggregate population gradually decreased and the number of free platelets increased in a time - dependent manner . fig4 depicts the time course of dissociation of platelet aggregates induced by plasma , humate - p , and emp . after platelet aggregates were induced for 10 min , the mixtures were diluted with pbs ( 1 : 20 ) to initiate dissociation . the time for 50 % dissociation for plasma , humate - p and emp were about 15 , 25 and 60 minutes respectively . these results demonstrate that platelet aggregates induced by emp are more stable than those induced by plasma or humate - p . we postulated that the greater stability of the aggregates formed by emp may be due to ( i ) the presence of very large multimers of vwf on emp and / or ( ii ) the presence of other adhesion molecules contributing to cross - linking between emp and platelets . effects of ttp plasma on ristocetin induced platelet aggregate formation . because abnormal degree of vwf multimerization has been implicated in ttp ( 37 , 38 ), we investigated plasmas from four ttp patients in acute ( a ) and remission ( r ) phases , compared to normal pooled plasma . as shown in table 2 , ttp patients exhibited significantly increased ristocetin - induced platelet aggregation , in both acute and remission states . ppp ( 4 %) from four different ttp patients in acute ( a ) and remission ( r ) phases and pooled control plasma were incubated with platelets and ristocetin for 10 min , then the remaining free platelets were assayed by flow cytometry . the platelet aggregate formation by ttp plasma in acute or remission phase was compared to the control group , mean ± s . d . * indicates p & lt ; 0 . 05 comparing ttp in acute phase or ttp in remission phase vs . the “ control ” group . the platelet aggregates produced by ttp plasma were also markedly more stable than with normal plasma . as shown in fig5 , the platelet aggregates with ttp plasma were much more resistant to dissociation after 1 : 20 dilution than normal plasma , and this was seen in both acute ( exacerbation ) and remission phases . filtration of the ttp plasma through 0 . 1 um to remove mp of size ≧ 0 . 1 um facilitated dissociation partially . the time course of dissociation with ttp plasma of acute phase plasma was similar to that of emp ( fig4 ). these results indicate that emp - bound vwf in ttp plasma may contribute in part to stabilizing platelet aggregates . application of emp - bound vwf to vwd plasma as shown in fig6 , plasma from four vwd patients was used to evaluate their vwf - dependent platelet aggregating activities . plasma from vwd patients showed very weak platelet aggregating activity . however , this activity increased dramatically after addition of humate - p ( 0 . 1 u / ml , final conc .). the figure also shows that addition of emp ( 5 × 10 6 / ml , final conc .) to the vwd patient plasma in vitro restored partially the aggregation activity of the vwd plasma . a synergistic effect of emp and humate - p combined induces strong platelet aggregate formation . multimer analysis of emp - bound vwf compared to vwf from normal plasma ttp plasma , and humate - p . we postulated that the observed effects of emp on ristocetin - induced platelet aggregation could be due to the presence of unusually large vwf multimers ( ulvwf ) on emp . as shown in fig7 , multimer analysis confirms that emp - bound vwf multimers ( lane 4 ) are larger than those from normal plasma ( lane 3 ), and even larger than those from humate - p ( lane 5 ), or plasma from a ttp patients whose plasma exhibited ulvwf ( lane 2 ). we also noticed that vwf multimers from a vwd type i patient contain very few bands ( lane 1 ). we have noticed that the vwf multimer bands from emp sample are not clearly separated from each other as observed with soluble vwf . one possible explanation for this is that membrane bound vwf multimers may be tightly bound with certain membrane phospholipids that are not fully dissociated by sds , which may cause more diffuse bands . use of emp composition to decrease bleeding time in vivo . in order to demonstrate in vivo efficacy of the emp , adult fischer rats were divided into three groups . group 1 served as normal controls . groups 2 and 3 were injected intraperitoneally with a single dose of cyclophosphamide ( ctx , 75 mg / kg ) to induce thrombocytopenia . after 4 - 5 days , when the platelet count was reduced to less than 5 × 10 5 / μl in the treated groups , the bleeding time was measured by clipping the tail 2 mm from the tip under anesthesia . prior to testing , group 3 was injected intravenously with 2 × 10 8 emp in 0 . 5 ml pbs two minutes prior to tail clipping . as shown in fig8 , in normal controls , bleeding time was less than one minute . group 2 , which received ctx only , had an average bleeding time of more than 800 seconds . group 3 , which received ctx + emp , had a greatly reduced bleeding time ( less than 200 seconds ) compared to group 2 . these results demonstrate the hemostatic potency of emp in vivo . clinical observations on atypical non bleeding itp patients . we have recently observed a limited number of itp patients who are highly unusual in their absence of bleeding symptoms ( asymptomatic ) in spite of severe thrombocytopenias . their platelet counts were 10 . 000 or less in most of time in their clinical courses of itp over 30 years but they never experienced major bleeding and enjoy fairly normal life . investigation on these patients revealed that exhibit exceptionally high levels of rmp ( two shown , a , b ). see fig9 . they exhibit no other abnormal feature to account for absence of bleeding . we attribute their asymptomatic features to the protective effect of their high rmp , as also supported by data below . two patients were identified with severe itp ( platelet counts 10 , 000 or less ) who nevertheless lived normally for over 40 years , with not a single episode of major bleeding . careful studies of these patients revealed that both had extremely high levels of red cell microparticles ( rmp ), compared to other itp patients who tend to bleed ( 49 , 50 ). ( see fig9 ). clinical observation on these two patients ( case studies a and b below ) suggested to us that rmp are hemostatically active and that the high level of rmp could account for the unusual absence of bleeding in these patients . case study ( a ). patient a developed itp at age 4 yr , when her mother noted easy bruising . upon subsequent evaluation a diagnosis of itp was made . she underwent splenectomy after alternative therapies failed . remission after splenectomy lasted one year ; itp relapsed shortly after polio vaccination . her itp responded only transiently to high dose glucocorticoids , iv gammaglobulin and not at all to other measures ( vinca alkaloids , danazol , colchicines , prosorba column , winrho , etc .). over the course of 47 yr of chronic itp , her platelets stayed around 10 , 000 / ul . she manifested bruises upon minor trauma , occasionally petechiae , but seldom suffered from mucosal bleeding and never experienced a major bleeding event requiring blood transfusion . she experienced heavy menstrual bleeding temporarily requiring prescription of birth control pills , which controlled the bleeding . she now lives a normal life as an active wife and mother . she also suffered frequent migraines requiring frequent parenteral pain medication . because of severe thrombocytopenia , multiple cat scans and mri were performed for fear of cns bleeding but were negative . she never had this complication . clinical results : platelets 7 , 000 / ul , hemoglobin 13 . 3 , hematocrit 39 . 1 %, wbc 9 . 3 with normal differential . blood chemistries including ldh were all normal . pt , aptt and other coagulation tests were all within normal limits . ana , c3 , c4 were normal . antiphospholipid antibodies and lupus anticoagulant were negative . antibodies against platelet glycoprotein iib / iiia and ib / ix , measured by platelet associated igg characterization assays , “ paica ” ( 52 ), were strongly positive , supporting diagnosis of itp . cell - derived microparticles from platelets ( pmp ), leukocytes ( lmp ), endothelium ( emp ) were all normal , but rmp were markedly elevated at 4 . 639 / ul , about 3 - fold higher than normal controls and more than twice as high as limit for itp ( normal mean = 1500 / ul ; normal itp mean = 2200 / ul ). case study ( b ). patient b was found to have itp in infancy and underwent splenectomy at age 4 months , giving partial remission . her itp responded well to glucocorticoids but she tolerated them poorly . platelets remained usually below 10 , 000 without treatment . she had heavy menstruation but this did not interfere with her normal activities . during the course of her chronic itp , now more than 50 years , she carried out normal activities and employment , never experiencing a major bleeding episode nor requiring transfusion . she delivered 2 children without unusual bleeding , and tolerated ankle and knee surgeries . although she bruised easily on minor trauma and had a few petechiae on careful examination , she never experienced prolonged mucosal bleedings such as nose or gum bleeding , or gi or gu bleedings . clinical results : platelets at 9 , 000 / ul , hemoglobin 13 . 7 , hematocrit 44 %, wbc 10 , 300 with normal differential . blood chemistries were all within normal limits , as were blood coagulation studies . the patient &# 39 ; s antinuclear antibody ( ana ) test was negative . antiphospholipid antibodies and lupus anticoagulant were all negative . antibodies to platelet glycoproteins iib / iiia and ib / ix were all strongly positive , consistent with itp . analysis of her coagulation studies , assays of cell derived microparticles from platelet ( pmp ), leukocytes ( lmp ), endothelial cells ( emp ) were all within normal limits . her only abnormality was markedly elevated rmp at 4 , 438 / ul , roughly 2 times higher than usual itp patients . the study on these two patients with severe chronic itp of over 50 years duration revealed that rmp were markedly elevated ( unusual compared to other itp patients ) but microparticles derived from other cells such as platelet microparticles ( pmp ), leukocyte microparticles ( lmp ) and other laboratory studies were not . these findings support the conclusion that rmp played a key role in protecting them from life - threatening bleeding episodes , since all other laboratory findings were comparable to other itp patients . in vitro laboratory data supporting that rmp are effective hemostatic agents . we developed several methods to generate rmp in vitro . after generation of rmp , expression of tissue factor ( tf ) and clotting factor viii ( fviii ) were assayed by immunological methods . in addition , we performed functional tests to determine if rmp can shorten clotting time ( 50 ). we prepared rmp which naturally express tf , fviii , as described below and shown in fig1 . tf - expressing rmp can be used as the basic or universal hemostatic agent in prevention of bleeding in patients with bleeding disorders as well as healthy people at risk of bleeding in situations like surgical or diagnostic procedures . rmp were prepared from freshly drawn normal blood using several methods . briefly : ( a ) ionophore method . washed rbc were exposed to calcium ionophore in the presence of added calcium . ( b ) osmotic shock method . washed rbc were exposed to hypotonic saline ( ⅓ of isotonic ). ( c ) ultrasonic method . washed rbc were exposed to brief bursts from ultrasound probe ( sonication ). ( d ) anti - d method with / without complement . the starting material was fresh rbc washed 3 times with isotonic saline as usual . two levels of anti - d ( winrho ) were tested , 10 u and 50 u per ml of original blood , added at 50 % ht , then incubated with gentle shaking for 50 min . then intact rbc were removed by low - speed centrifugation and rmp were pelleted by high - speed centrifugation , as usual , and resuspended for flow cytometry . flow cytometry . rmp were identified by fluorescent monoclonal antibody ( mab ) against rbc marker glycophorin a . also measured on rmp by mab was tissue factor ( tf ) and clotting factor viii ( fviii ). fluorescent annexin v ( anv ) was employed to measure exposure of procoagulant phosphotidyl serine ( ps ), and fitc - labeled lectin , ulex europaeus ( ulex ) was used to give an estimate of total mp ( 50 ). flow cytometry revealed that all three methods ( a , b , c ) yielded abundant rmp . of special interest is that they express weakly but significantly positive for tissue factor ( tf ), a potent initiator of coagulation ( active at very low levels ). fviii was identified at similar levels . ( see fig1 ). representative results of nine experiments by three methods ( ionophore , sonication , and anti - d ). fig1 shows total rmp as defined by number of particles positive for glyocphorin and that this fraction is positive for tf , fviii and annv . notice that ps exposure ( reflected in anv positives ) is usually low . this suggests a good half - life in circulation because ps is a trigger for phagocytosis . procoagulant activity assay of rmp . the rmp from 1 . 5 ml of rmp prepared in standard way were sedimented by centrifuging 15 min at 8 , 000 × g ( eppendorf microfuge ) and the supernatant removed . then standard ( normal ) plasma was added , the rmp resuspended . to assess procoagulant activity , rmp is added to the mixture and the recalcification time was measured by adding calcium ( 2 mm ). clot time was measured manually . the results ( shown in fig1 ) demonstrate that rmp have significant procoagulant activity , as further detailed below . there are at least two known pathways of blood coagulation . one is an “ intrinsic ” pathway that is completely inhibited by corn trypsin inhibitor , and a tf mediated “ extrinsic ” pathway . without being limited to any particular pathway , the inventors surmise that the because the procoagulant activity of rmp persists in the presence of corn trypsin inhibitor , rmp procoagulant activity is likely due to tf mediation . not shown are experiments which included corn trypsin inhibitor ( which abolishes non - tf mediated procoagulant activity ). procoagulant activity of rmp . fig1 shows recalcification clotting time in minutes , mean of replicates +/− standard deviation using the method described above . as can be seen , there is a marked shortening of clotting time in the presence of rmp . surprisingly , the rmp were more effective in this experiment than a similar quantity of leukocyte - derived mp ( lmp ) but the difference was not significant . similar results were confirmed in the presence of corn trypsin inhibitor ( 50 ). animal in vivo data demonstrating that rmp are hemostatically active . we studied the efficacy of red blood cell microparticles ( rmp ) in a dose - dependent manner in adult fischer rats . the animals were randomized into 4 groups . group 1 served as normal controls . groups 2 , 3 and 4 were injected i . p . with a single dose of cyclophosphamide ( 75 mg / kg ) to induce thrombocytopenia . at 5 days , when the platelet count was reduced to less than 5 × 10 5 / microliters in the treated groups , the bleeding time was measured by clipping the tail 2 mm from the tip under anesthesia . two minutes prior to testing bleeding time , groups 3 and 4 were injected with 1 × 10 7 and 1 × 10 8 rmp respectively . rmp were prepared essentially as follows : observing strictly sterile techniques throughout , whole rbc from freshly drawn citrate - treated blood was washed twice with 10 volumes of isotonic saline and then was suspended to 17 % hematocrit , then was exposed to ultrasonic burst ( cole - parmer , model 4710 , ultrasonic homogenizer , fitted with small probe ) for 1 second . large debris was removed by low - speed centrifugation ( 8 min , 200 × g in beckman clinical centrifuge , then the supernatent was centrifuged for 15 min at 8 , 000 × g in eppendorf microfuge ( in 1 . 5 ml polypropylene tubes ) and the burgundy - colored supernatent was removed . the small pellet of rmp was suspended in a small volume , counted by fitc - labeled ulex europaeus , and then diluted to the concentration indicated prior to injection in the experimental animal the results are shown in fig1 . in normal controls , bleeding time was less than one minute . group 2 , after thrombocytopenia induction and which received cyclophosphamide only , had a bleeding time of over 700 seconds . group 3 , which received 1 × 10 7 rmp after thrombocytopenia induction , had a greatly reduced bleeding time compared to group 2 . a dose - dependent effect was observed of rmp administration , with a still greater decrease in bleeding time observed in animals treated with 1 × 10 8 rmp . conjugation of clotting factors or adhesions to rmp . rmp can be biochemically modified to augment their hemostatic activity for some applications . for example , rmp may be modified by ultrasonic incorporation of polyethylene glycol ( peg ) since it has been shown that pegylated liposomes then avidly but non - covalently adsorb both fviii and vwf ( 55 ). in another approach , rmp may be modified by the covalent addition of rgd peptide [ 53 , 62 ]. methods for conjugation of specific proteins or peptides to phospholipids ( pl ) vesicles are well - known in the art , see in particular the comprehensive text by greg t . hermanson , bioconjugate techniques ( 54 ). since cell derived microparticles are essentially pl vesicles , it is reasonably expected that the same methods are applicable . it is envisioned that the agent to be conjugated to rmp ( e . g . rgd peptide ) can be pre - activated intermediates and stored in sterile lyophilized form ( for example , see ( 54 ), ( page 236 ). when needed , it may be added to the patient rmp , resulting in protein - rmp conjugates after one hour incubation , then requiring only that the conjugated rmp be washed free of excess of reagents . references cited herein are hereby incorporated by reference and are listed below for convenience 1 . horstman l l , ahn y s . platelet microparticles : a wide - angle perspective ( review ). crit . rev oncol / hematol 1999 ; 30 : 111 - 142 . 2 . horstman l l , jy w , jimenez j j , ahn y s . endothelial microparticles as markers of endothelial dysfunction ( review ). frontiers in bioscience 2004 ; 9 : 1118 - 1135 . 3 . ahn y s . cell - derived microparticles : miniature envoys with many faces . j thromb and hemostasis . 3 : 884 - 887 , may , 2005 . 4 . dachary - prigent j d , freyssinet j - m , pasquet j - m , carron j - c , nurden a t . annexin v as a probe of aminophospholipid exposure and platelet membrane vesiculation : a flow cytometric study showing a role for free sulfhydryl groups . blood 1993 ; 81 : 2554 - 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