Proteolysis of plasma-derived factor V following its endocytosis by megakaryocytes forms the platelet-derived factor V/Va pool.

BACKGROUND: Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa on the activated platelet surface. While the plasma-derived procofactor, FV, must be proteolytically activated by α-thrombin to FVa to function in prothrombinase, the platelet molecule is released from α-granules in a partially activated state, obviating the need for proteolytic activation. OBJECTIVES: The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes, plasma-derived FV is proteolytically processed to form the platelet-derived pool. METHODS & RESULTS: Subsequent to FV endocytosis, a time-dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS-PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/Va present in a platelet lysate as well as to the α-thrombin-activated FVa heavy chain and light chain, and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti-FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet-derived FV/Va. CONCLUSIONS: Taken together, these observations suggest that prior to its packaging in α-granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool.

A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes.

BACKGROUND: Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique platelet-derived FV pool. OBJECTIVE: The role of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), or a related family member, in FV endocytosis by megakaryocytes was examined because of its known interactions with other proteins involved in hemostasis. METHODS: LRP-1 expression by megakaryocytes and its functional role in FV endocytosis was confirmed using reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies. FV binding to megakaryocytes was performed under Ca(2+)-free conditions to quantify binding in the absence of endocytosis. RESULTS AND CONCLUSION: Cell surface expression of LRP-1 by CD34+ ex vivo-derived megakaryocytes and the megakaryocyte-like cell line CMK was confirmed using anti-LRP-1 antibodies and was consistent with the detection of LRP-1 message in these cells. All cells capable of endocytosing FV expressed LRP-1. Anti-LRP-1 antibodies and receptor-associated protein (RAP), a known antagonist of LDL receptor family members, displaced only 50% of the [(125)I]FV bound to megakaryocytes. FV binding to megakaryocytes showed positive cooperativity (Hill coefficient = 1.92 +/- 0.18) that was substantially reduced in the presence of RAP (1.47 +/- 0.26). As FV endocytosis is specific to this cofactor, a model is hypothesized where FV binding to a specific receptor facilitates binding and endocytosis of a second FV molecule by LRP-1, or a related family member. These combined observations describe a unique role for LRP-1 in endocytosis of a coagulation protein trafficked to alpha-granules and not destined for lysosomal degradation.

Megakaryocytes endocytose and subsequently modify human factor V in vivo to form the entire pool of a unique platelet-derived cofactor.

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential cofactor in thrombin production catalyzed by the prothrombinase complex. Plasma-derived factor V (FV) is synthesized in the liver. The source(s) of the platelet-derived cofactor remains in question. We identified a patient homozygous for the FV(Leiden) mutation, who received a liver transplant from a homozygous wild-type FV donor. Eighteen days post-transplant, phenotypic analysis of the patient's platelet-derived FV indicated that the platelets were acquiring wild-type FV, consistent with the temporal differentiation of megakaryocytes and subsequent platelet production. Nine months post-transplant, the platelet-derived FV pool consisted entirely of wild-type FV. Consequently, megakaryocyte endocytosis of plasma-derived FV must account for the entire platelet-derived pool, because blood-borne platelets cannot bind or endocytose FV. Subsequent to this endocytic process, the patient's platelet-derived FV was cleaved to a partially active cofactor, and rendered resistant to phosphorylation catalyzed by a platelet-associated kinase, and hence less susceptible to activated protein C-catalyzed inactivation. These data provide the first in vivo demonstration of an endocytosed plasma protein undergoing intracellular modifications that alter its function. This process results in the sequestration of active FVa within the platelet compartment, poised for immediate action subsequent to release from platelets at a site of injury.

Endocytosis of plasma-derived factor V by megakaryocytes occurs via a clathrin-dependent, specific membrane binding event.

Megakaryocytes were analyzed for their ability to endocytose factor V to define the cellular mechanisms regulating this process. In contrast to fibrinogen, factor V was endocytosed by megakaryocytes derived from CD34(+) cells or megakaryocyte-like cell lines, but not by platelets. CD41(+)ex vivo-derived megakaryocytes endocytosed factor V, as did subpopulations of the megakaryocyte-like cells MEG-01, and CMK. Similar observations were made for fibrinogen. Phorbol diester-induced megakaryocytic differentiation of the cell lines resulted in a substantial increase in endocytosis of both proteins as compared to untreated cells that did not merely reflect their disparate plasma concentrations. Factor IX, which does not associate with platelets or megakaryocytes, was not endocytosed by any of the cells examined. Endocytosis of factor V by megakaryocytes proceeds through a specific and independent mechanism as CHRF-288 cells endocytosed fibrinogen but not factor V, and the presence of other plasma proteins had no effect on the endocytosis of factor V by MEG-01 cells. Furthermore, as the endocytosis of factor V was also demonstrated to occur through a clathrin-dependent mechanism, these combined data demonstrate that endocytosis of factor V by megakaryocytes occurs via a specific, independent, and most probably receptor-mediated, event.

Platelets, leukocytes, and coagulation.

Considerable data now support the hypothesis that platelets actively regulate the propagation of coagulation by (1) expressing specific, high-affinity receptors for coagulation proteases, zymogens, and cofactors; (2) protecting the bound coagulation enzymes from inactivation/inhibition; (3) restricting coagulant activity to the site of vascular injury; and (4) amplifying the initiating stimulus to lead to explosive thrombin generation. Thrombin generation is sustained at the site of vascular injury by the recruitment of circulating monocytes and neutrophils to the growing thrombus via the interaction of PSGL-1, which is constitutively expressed by leukocytes, with P-selectin, which is expressed by activated platelets. Unique among cells, monocytes can provide the appropriate membrane surface for the assembly and function of all the coagulation complexes required for tissue factor-initiated thrombin production. More studies are required to further delineate the roles of neutrophils and lymphocytes in the procoagulant response. This review will discuss the recent investigations and controversies regarding the various mechanisms by which platelets and leukocytes function in, and regulate, thrombin generation.

Platelet regulation of thrombin generation in cardiovascular disease.

Platelets are intimately involved in the events leading to cardiac ischemia through their release of bioactive substances, aggregation, and support of procoagulant reactions at sites of atherosclerotic plaque formation and rupture. This review article will focus on what is currently known about the regulation of thrombin generation on the surface of activated platelets, and how it relates to thrombus formation.

The platelet high affinity binding site for thrombin mimics hirudin, modulates thrombin-induced platelet activation, and is distinct from the glycoprotein Ib-IX-V complex.

The platelet high affinity binding site for thrombin appears to be described by a classical receptor-ligand interaction that is distinct from the platelet thrombin receptor/substrate, PAR-1. However, the identification and function of the high affinity binding site with respect to its physiological importance have continued to elude investigators. Prior studies using two mutant thrombins suggested that thrombin interaction with the platelet high affinity binding site is mediated through an extensive portion of the thrombin molecule involving residues within the substrate binding pocket and the anion binding exosite (Leong, L., Henriksen, R. A., Kermode, J. C., Rittenhouse, S. E., and Tracy, P. B. (1992) Biochemistry 31, 2567-2575) and may mimic a thrombin-hirudin interaction. To test this hypothesis, an anti-hirudin peptide antibody (anti-hirpeptide Ab) was raised against a peptide mimicking the COOH terminus of hirudin. The Ab recognized adherent platelets and those in suspension as determined by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. 125I-Thrombin binding to platelets was inhibited in the presence of the anti-hirpeptide Ab in a dose-dependent manner with maximal inhibition >90%. Analyses of data from binding studies of 125I-thrombin to platelets at a fixed Ab concentration indicated that the anti-hirpeptide Ab inhibited the high affinity binding interaction exclusively. In addition, thrombin-induced increases in platelet [Ca2+]i were enhanced by blocking the high affinity binding site with the Ab due to redistribution of the agonist to PAR-1. Thrombin Quick I-induced platelet calcium mobilization was unaffected by the presence of the Ab, consistent with the inability of thrombin Quick I to bind to the high affinity site. Even though glycoprotein (GP) Ib contains a hirudin-like region within the alpha subunit, the postulated high affinity binding site, direct binding of 125I-thrombin could not be demonstrated to transfected Chinese hamster ovary and L cells expressing the GP Ib-IX-V complex. Furthermore, an anti-GP Ib Ab, raised to the peptide region proposed as the thrombin high affinity site, did not enhance thrombin-induced platelet calcium mobilization. The anti-hirpeptide Ab recognized a population of platelet membrane proteins distinct from PAR-1 and GP Ib by three-color immunofluorescence using confocal microscopy. These combined studies demonstrate that the high affinity binding site for thrombin is a unique platelet protein distinct from GP Ib which modulates the effective thrombin concentration localized at the human platelet surface.

Secretable human platelet-derived factor V originates from the plasma pool.

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into alpha-granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed.

Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding.

The single-chain procofactor factor V is cleaved by thrombin (FVaIIa) at Arg709, Arg1018, and Arg1545 and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/HNE. The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa. Significant differences in cofactor function were observed between the two assay systems. Whereas FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus, cleavage at Arg1545 optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.

Acute coronary syndromes: 2. Antiplatelet agents.

Therapies with novel antiplatelet agents and anticoagulants are the focus of current research. When used separately or in combination, these agents prevent generation of thrombin by activated platelets. The new therapies, in conjunction with judicious use of fibrinolytic drugs and mechanical interventions, are revolutionizing the management of patients with acute coronary syndromes.

Platelet-derived factor Va/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C.

We investigated the role of the thrombin-activated platelet in modulating the rate and extent of activated protein C (APC)-catalyzed inactivation of platelet-derived factor Va and factor VaLeiden. Platelet-derived factor Va and factor VaLeiden were inactivated by APC at near identical rates; however, complete inactivation of the cofactors was never achieved. Greater residual cofactor activity remained when using thrombin-activated platelets compared with that observed with synthetic phospholipid vesicles and platelet-derived microparticles, suggesting that thrombin-activated platelets protect the cofactors from APC-catalyzed inactivation. This apparent protection was not due to (1) an insufficient number of membrane binding sites for APC or factor Va; (2) the destruction of these sites; or (3) the presence of a platelet-associated APC inhibitor. Results from a plasma-based clotting assay (with or without APC) with platelets or PCPS vesicles added to induce clot formation indicated that, even in the presence of high concentrations of APC, platelets offered protection of the cofactor by delaying cleavage at Arg506. This resulted in incomplete proteolysis of the heavy chain, suggesting that platelets can also protect plasma-derived factor Va from APC-catalyzed inactivation. However, additional experiments indicated that the plasma-derived cofactor, bound to thrombin-activated platelets, was completely inactivated by APC, suggesting that the plasma and platelet-derived cofactor pools represent different substrates for APC. Collectively, these results indicate that platelets sustain procoagulant events by providing a membrane surface that delays cofactor inactivation and by releasing a cofactor molecule that displays an APC resistant phenotype. Thus, at sites of arterial injury, the factor VLeiden mutation may not as readily predict arterial thrombosis, because the normal and variant platelet-derived cofactors are equally resistant to APC at the activated platelet surface.

Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles.

This report describes the expression, purification, and characterization of a series of recombinant factor Xa variants bearing aspartate substitutions for each of the glutamate residues which normally undergo gamma-carboxylation. Factor X was expressed in human embryonic kidney cells and purified from conditioned media by immunoaffinity and hydroxylapatite chromatography. Factor X was activated with Russell's viper venom factor X activator, and single-chain unactivated factor X was removed from activated factor X by size-exclusion chromatography. Recombinant wild-type factor Xa had normal activity in a clotting assay, and mutants with aspartate substitutions for glas residues 16, 26, and 29 had no detectable clotting activity. In purified component assays, these gla variants had essentially no detectable activity in the prothrombinase complex assembled on synthetic phospholipid vesicles but had significant activity when the prothrombinase was assembled on thrombin-activated platelets. In addition, the gla 32 variant had normal activity in the platelet prothrombinase but diminished activity in prothrombinase assembled on synthetic PSPC vesicles. These differences were not accounted for by the total phospholipid composition of the thrombin-activated platelet membrane. We have produced fully active recombinant human factor Xa and demonstrated that gla residues 16, 26, and 29 are critical for normal activity of factor Xa. More importantly, this study provides an extensive characterization of macromolecular enzyme complex formation with gla variants of a vitamin K-dependent coagulation protein and provides evidence that prothrombinase complex assembly on thrombin-activated platelets is not equivalent to assembly on synthetic phospholipid vesicles. The data suggest that thrombin-activated platelets possess some element(s) (other than 30% phosphatidyl serine or factor Va), presumably either protein or phospholipid, that serves as a component of the factor Xa binding site.

Acute coronary syndromes: 1. The platelet's role.

Platelets play key physiologic roles in the hemostatic response to vascular injury. But thrombi that form in the absence of vessel wall damage may either disrupt or completely block blood flow. A better understanding of platelet pathophysiology promises to lead to improved therapy of myocardial infarction, unstable angina, and related syndromes.

Adenosine diphosphate-induced platelet activation inhibited by magnesium in a dose-dependent manner.

OBJECTIVE: To examine the hypothesis that magnesium inhibits platelet activation at concentrations equivalent to therapeutic levels. METHODS: Fifteen subjects were enrolled: five healthy, female donors with regular, spontaneous menstrual cycles; five women with uncomplicated third-trimester pregnancies; and five preeclamptic subjects before magnesium therapy. Anticoagulated whole blood was added to tubes containing 0.5 micromol/L adenosine diphosphate (to activate platelets), HP1-1D (activation-independent platelet antibody), CD62 antibody and CD63 antibody (activation-dependent platelet antibodies), and magnesium sulfate in increasing concentrations (2-100 mg/dL). The percentage of activated platelets (CD62 or CD63 positive) was determined using three-color flow cytometric analysis. Data were analyzed using the Student t test, repeated measures analysis of variance, two-way analysis of variance, and Student-Newman-Keuls for pairwise comparison in appropriate cases. P < .05 was considered significant. RESULTS: Adenosine diphosphate-induced platelet activation was inhibited with increasing magnesium concentration in all subjects (P < .001). Significant inhibition of CD62 and CD63 expression first occurred at a magnesium concentration of 4 mg/dL in the normal pregnant group (P < .05), at 6 mg/dL in the preeclamptic group (P < .05), and at 8 mg/dL in the nonpregnant group (P < .05). CONCLUSION: Magnesium inhibits adenosine diphosphate-induced platelet activation in a dose-dependent manner. This effect initially attains statistical significance at concentrations equivalent to therapeutic levels.

Plasma lipoproteins support prothrombinase and other procoagulant enzymatic complexes.

The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 micromol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 micromol/L phospholipid) yielded rates of 2 to 8 nmol Flla x L(-1) x s(-1) in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (< or =1.0 nmol FIIa x L(-1) x s[-1]). For comparison, VLDL at 2 mmol/L triglyceride yielded approximately 50% the activity of 2X10(8) thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylsenne vesicles (approximately 50 nmol FIIa x L(-1) x s[-1]), the prothrombin Km values were similar, 0.8 and 0.5 micromol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 nmol/L FIIa x L(-1) x s[-1]). These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.

Both cellular and soluble forms of thrombomodulin inhibit fibrinolysis by potentiating the activation of thrombin-activable fibrinolysis inhibitor.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.

Differential effects of anticoagulants on the activation of platelets ex vivo.

BACKGROUND: Because activation of platelets and of the coagulation system are interdependent mediators of thrombosis, platelet activation was characterized in whole blood in the presence of anticoagulants used to assess platelet function in vitro or as treatment for patients with occlusive arterial disease. METHODS AND RESULTS: Blood was anticoagulated alone or in combination with citrate, ethylenediaminetetraacetatic acid, corn trypsin inhibitor (CTI, an inhibitor of activated factor XII), heparin, enoxaparin, recombinant tick anticoagulant peptide (rTAP), or recombinant hirudin. Platelet activation in response to adenosine diphosphate (ADP) or collagen was detected by assay of P-selectin on the platelet surface delineated by flow cytometry. Although minimal activation was seen without ADP, the fraction of platelets expressing P-selectin in response to ADP was greatest in blood anticoagulated with citrate compared with CTI and all other anticoagulants. ADP-induced platelet activation was greater in blood anticoagulated with heparin compared with an equipotent anti-Xa concentration of enoxaparin. More variable results were seen with collagen, but platelet activation in the presence of citrate was greater than that with CTI. CONCLUSIONS: Interpretation of assays of inhibition of platelet activation by potentially therapeutic agents in vitro requires consideration of the effects of anticoagulants used. In addition, anticoagulants other than standard heparin may potentiate efficacy of antiplatelet drugs.