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Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones in vivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity in vitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Background: Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements., Methods: Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] < 0.9, n = 11) or hyperfibrinolysis (LY30 > 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA)., Results: Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator-stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH., Conclusion: This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma., Level of Evidence: Prognostic/Epidemiological; Level III., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Coagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. We studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Circulating histone-DNA complexes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). Using calibrated automated thrombography, thrombin generation was altered in COVID-19 patient plasma samples. Despite having increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. While healthy control plasma samples did not generate measurable thrombin after 60 min, plasma samples from COVID-19+ patients formed thrombin (mean lag time ~20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. Elevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation may contribute to coagulopathy in COVID-19., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tissue factor (TF) is the most important trigger for the extrinsic coagulation pathway. TF, earlier denoted as thromboplastin, has always been a mystery since its discovery due to its abundant presence in most human tissues but not blood. The latter has been extensively studied in a vast quest for possible sources of blood-borne TF yielding many conflicting findings and confusing conclusions regarding the presence of TF mRNA, protein or functional procoagulant activity in virtually all blood cells. Platelets, in particular, have been heavily scrutinized by investigators eager to demonstrate expression of TF. However, some investigators including our own groups have not found evidence for TF in platelets. This article discusses notable reports and possible reasons for erroneous detection of platelet TF antigen and activity including artificially hyper-stimulated platelets, suboptimal purity of cell preparations, flaws in study design and/or choice of reagents.

Plasma- and platelet-derived factor Va are essential for thrombin generation catalyzed by the prothrombinase complex; however, several observations demonstrate that the platelet-derived cofactor, which is formed following megakaryocyte endocytosis and modification of the plasma procofactor, factor V, is more hemostatically relevant. Factor V endocytosis, as a function of megakaryocyte differentiation and proplatelet formation, was assessed by flow cytometry and microscopy in CD34 + hematopoietic progenitor cells isolated from human umbilical cord blood and cultured for 12 days in the presence of cytokines to induce ex vivo differentiation into megakaryocytes. Expression of an early marker of megakaryocyte differentiation, CD41, endocytosis of factor V, and the percentage of CD41 + cells that endocytosed factor V increased from days 6 to 12 of differentiation. In contrast, statistically significant decreases in expression of the stem cell marker, CD34, and in the percentage of CD34 + cells that endocytosed factor V were observed. A statistically significant increase in the expression of CD42b, a late marker of megakaryocyte differentiation, was also observed over time, such that by Day 12, all CD42b + cells endocytosed factor V and expressed CD41. This endocytosed factor V was trafficked to proplatelet extensions and was localized in a punctate pattern in the cytoplasm consistent with its storage in α-granules. In conclusion, loss of CD34 and expression of CD42b define cells capable of factor V endocytosis and trafficking to proplatelet extensions during differentiation of megakaryocytes ex vivo from progenitor cells isolated from umbilical cord blood., (© 2018 Wiley Periodicals, Inc.)

Chronic kidney disease (CKD) affects 30 million Americans and is associated with approximately a two-fold increased risk of venous thromboembolism (VTE). There is a graded increased risk of VTE across declining kidney function, as measured by estimated glomerular filtration rate (eGFR) and albuminuria. When patients with end-stage kidney disease (ESKD) experience VTE they are more likely than the general population to be hospitalized and they have a higher mortality. The incidence and consequences of VTE may also differ depending on the cause of kidney disease. In addition, kidney transplant patients with VTE are at a greater risk for death and graft loss than transplant patients without VTE. The reasons that patients with CKD are at increased risk of VTE are not well understood, but recent data suggest that factor VIII is a mediator. Factor VIII is an essential cofactor in the coagulation cascade and a strong risk factor for VTE in general. It is inversely correlated with eGFR and prospective studies demonstrate that factor VIII activity predicts incident CKD and rapid eGFR decline. The etiology of CKD may also influence factor VIII levels. This review summarizes the epidemiology VTE in CKD and reviews the biochemistry of factor VIII and determinants of its levels, including von Willebrand factor and ABO blood group. We explore mechanisms by which the complications of CKD might give rise to higher factor VIII and suggests future research directions to understand how factor VIII and CKD are linked., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Intact red blood cells (RBCs) appear to support thrombin generation in in vitro models of blood coagulation. During storage of RBC units, biochemical, structural, and physiological changes occur including alterations to RBC membranes and release of microparticles, which are collectively known as storage lesion. The clinical consequences of microparticle formation in RBC units are unclear. This study was performed to assess thrombin generation via the prothrombinase complex by washed RBCs and RBC-derived microparticles as a function of RBC unit age., Methods: Well-characterized kinetic and flow cytometric assays were used to quantify and characterize microparticles isolated from leukocyte-reduced RBC units during storage for 42 days under standard blood banking conditions., Results: Stored RBCs exhibited known features of storage lesion including decreasing pH, cell lysis, and release of microparticles demonstrated by scanning electron microscopy. The rate of thrombin formation by RBC units linearly increased during storage, with the microparticle fraction accounting for approximately 70% of the prothrombinase activity after 35 days. High-resolution flow cytometric analyses of microparticle isolates identified phosphatidylserine-positive RBC-derived microparticles; however, their numbers over time did not correlate with thrombin formation in that fraction., Conclusion: Red blood cell-derived microparticles capable of supporting prothrombinase function accumulate during storage, suggesting an increased potential of transfused units as they age to interact in unplanned ways with ongoing hemostatic processes in injured individuals, especially given the standard blood bank practice of using the oldest units available.

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbβ 3 , α v β 3 ), and purinergic receptors (P2Y 1 , P2Y 12 ), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble N -ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function. Although not required for normal platelet exocytosis or hemostasis, VAMP-3 -/- mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Other granule markers were unaffected. Direct experiments, both in vitro and in vivo, showed that loss of VAMP-3 led to a robust defect in uptake/storage of Fg in platelets and cultured megakaryocytes. Uptake of the fluid-phase marker, dextran, was only modestly affected. Time-dependent uptake and endocytic trafficking of Fg and dextran were followed using 3-dimensional-structured illumination microscopy. Dextran uptake was rapid compared with Fg, but both cargoes progressed through Rab4 + , Rab11 + , and von Willebrand factor (VWF) + compartments in wild-type platelets in a time-dependent manner. In VAMP-3 -/- platelets, the 2 cargoes showed limited colocalization with Rab4, Rab11, or VWF. Loss of VAMP-3 also affected some acute platelet functions, causing enhanced spreading on Fg and fibronectin and faster clot retraction compared with wild-type. In addition, the rate of Janus kinase 2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3 -/- platelets. Collectively, our studies show that platelets are capable of a range of endocytosis steps, with VAMP-3 being pivotal in these processes., (© 2017 by The American Society of Hematology.)

Differentiating megakaryocytes undergo a unique endomitotic cell cycle leading to large polyploidal cells, which fragment to generate platelets, blood cells important for normal hemostasis. Simultaneous assessment of DNA content and cellular proteins by flow cytometry is a useful tool to study megakaryocyte differentiation and to define expression of proteins important for megakaryocyte development and platelet formation. The usefulness of zinc salt-based fixation (ZBF), a non-crosslinking method of cell fixation that permits downstream analysis of nucleic acids (Jensen et al., Cytometry A 2010;77A:798-804), in flow cytometric analysis of megakaryocyte ploidy in conjunction with extracellular and intracellular proteins was assessed. ZBF of a megakaryocyte-like cell line resulted in preservation of proteins similar to paraformaldehyde fixation, and preservation of DNA content in a manner similar to methanol fixation. This is highlighted by experiments in which polyploidal megakaryocytes were analyzed simultaneously for endocytosis of a fluorescently-labeled, endocytosed labile protein or expression of a cell surface integrin and DNA content. These studies demonstrate that ZBF will be a valuable tool to study the molecular events leading to platelet formation. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function., (© 2016 by The American Society of Hematology.)

Factor Va serves as the nonenzymatic protein cofactor for the prothrombinase complex, which converts prothrombin to thrombin in the events leading to formation of a hemostatic plug. Several observations support the concept that platelet-derived factor V/Va is physically and functionally distinct and plays a more important role in thrombin generation at sites of vascular injury as compared to its plasma counterpart. Platelet-derived factor V/Va is generated following endocytosis of the plasma-derived molecule by the platelet precursor cells, megakaryocytes, via a two receptor system consisting of low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) and an unidentified specific "binding site". More recently, it was suggested that a cell surface-expressed β-galactoside binding protein, galectin-8, was involved in factor V endocytosis. Endocytosed factor V is trafficked through the cell and retailored prior to its storage in α-granules. Given the essential role of platelet-derived factor Va in clot formation, understanding the cellular and molecular mechanisms that regulate how platelets acquire this molecule will be important for the treatment of excessive bleeding or clotting., (© 2015 Wiley Periodicals, Inc.)

Whole genome sequencing of an individual completely devoid of plasma- and platelet-derived factor V (FV) identified 167 variants in his F5 gene including previously identified and damaging missense mutations at rs6027 and Leu90Ser. Because the administration of fresh frozen plasma (FFP) prevents gastrointestinal bleeding in this individual, its effects on his plasma- and platelet-derived FV concentrations were assessed. The patient's plasma FV levels peaked by 2 hours following FFP administration and were undetectable 96 hours later. In contrast, increased platelet-derived FV/Va concentrations were observed within 6 hours, peaked at 24 hours, decreased slowly over 7 days, and originated from megakaryocyte endocytosis and intracellular processing of plasma FV. Ten days after transfusion, no thrombin was generated in a tissue factor-initiated whole blood clotting assay unless exogenous FV was added, consistent with the complete absence of plasma FV. In marked contrast, release of the patient's platelet-derived FV/Va (7% of normal) following platelet activation resulted in robust thrombin generation, similar to that in an individual with normal plasma- and platelet-derived FV concentrations. Thus, total FV deficiency can be corrected by plasma administration, which partially repletes and sustains the platelet cofactor pool, thereby highlighting the critical role of platelet-derived FV/Va in ensuring hemostatic competence., (© 2015 by The American Society of Hematology.)

While platelets are well known to play a central role in hemostasis and thrombosis, there is emerging experimental evidence to suggest that they also mediate tumor cell growth, dissemination, and angiogenesis. An increase in platelet number (thrombocytosis) and activity is seen in patients with a wide spectrum of malignancies, and the former is correlated with a decrease in overall survival and poorer prognosis. Preclinical data suggest that circulating tumor cell partnerships with platelets in the blood facilitate tumor metastases through direct interactions and secreted bioactive proteins. Platelets form aggregates with tumor cells, thereby protecting them from host immune surveillance through physical shielding and induction of "platelet mimicry." There is also laboratory evidence to suggest that activated platelets interact with cancer cells within the tumor microenvironment through paracrine signaling and direct contact, thereby promoting tumor cell growth and survival. For example, platelets release mediators of both tumor angiogenesis and osteoclast resorption. The interplay between platelets and tumor cells is complex and bidirectional with involvement of multiple other components within the tumor microenvironment, including immune cells, endothelial cells, and the extracellular matrix. We review the role of platelets in tumor progression, emphasizing the opportunity these interactions afford to target platelets and platelet function to improve patient outcomes in the cancer prevention and treatment setting., (© 2013 Wiley Periodicals, Inc.)

Background: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms., Methods: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results., Results: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography., Conclusion and Significance: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (© 2013 International Society on Thrombosis and Haemostasis.)

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30-40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.

Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin cell adhesion molecule family. Recently, we identified CADM1 to be a novel risk factor for venous thrombosis in a large, protein C deficient, thrombophilic family and showed, for the first time, the expression of CADM1 in endothelial cells (Hasstedt et al. in Blood 114:3084-3091, 2009). To further investigate its role in venous thrombosis, as well as other vasculopathies, we undertook a systematic confocal microscopic investigation for the presence of CADM1 in the vasculature of 28 different human tissues. Paraffin embedded tissue sections were dual immunostained with an antibody against CADM1, together with an antibody against either von Willebrand factor (to identify endothelial cells), or α-smooth muscle actin (to identify smooth muscle cells). The results showed that CADM1 was ubiquitously present in endothelial cells and smooth muscle cells in the vasculature from all 28 tissues, though its representation in the various classes of vessels was tissue dependent.

Objective: The goal of this study was to define and characterize the subpopulation of platelets capable of regulating the functional interactions of factors Va (FVa) and Xa (FXa) on the thrombin-activated platelet surface., Methods and Results: Flow cytometric analyses were used to define and characterize platelet subpopulations. At a concentration of thrombin known to elicit maximal platelet activation, platelet-derived FVa release, and prothrombinase assembly/function, only a subpopulation of platelets was positive for FVa and FXa binding. An additional subpopulation bound lower levels of FVa but little, if any, FXa. Fluorescence microscopy analyses confirmed these data. Phenotypically, platelets capable of binding FXa were more highly reticulated and demonstrated significantly increased expression of several key adhesion molecules, including P-selectin, glycoprotein Ibα, and integrins α(IIb) and β(3). This platelet subpopulation was also defined by the expression of a nondissociable, membrane-bound pool of functional platelet-derived FVa, which made up ≈35% to 50% of the total membrane-bound cofactor., Conclusions: The ability of activated platelets to support thrombin generation is defined by a subpopulation of platelets expressing a nondissociable pool of platelet-derived FVa and increased adhesive receptor density. This subpopulation is hypothesized to play a significant role in regulating both normal hemostasis and pathological thrombus formation because the adherent properties of platelets and their ability to mount and sustain a procoagulant response are crucial steps in both of these processes.

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.

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

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.

Objective: Preeclampsia is associated with increased platelet activation, increased sympathetic activity, and decreased plasma volume. We sought to estimate the relationship of plasma volume, sympathetic activity, or both to platelet activation in nonpregnant nulligravid women., Methods: We studied 37 healthy nulligravid subjects during the follicular phase of the menstrual cycle. After intravenous access was obtained, subjects rested in the supine position for 15 minutes. Blood was drawn without venous constriction for measurement of plasma catecholamines (epinephrine and norepinephrine) and complete blood count. Antigenic markers of platelet activation, CD63 and CD61-CD14 (platelet-monocyte aggregates), were measured with flow cytometry. Plasma volume was estimated in the supine position by using Evans blue dye and is expressed in milliliters and corrected for body mass index (BMI). We compared data from the lowest plasma volume/BMI quartile with the 2 middle quartiles combined and with the upper quartile. Data are expressed as mean +/- standard deviation. P <.05 was considered significant., Results: Subjects were aged 26.5 +/- 5.0 years, BMI was 24.0 +/- 3.0 kg/m(2), and plasma volume was 2,685 +/- 429 mL. We identified no significant relationship of platelet concentration to plasma volume/BMI between quartile groups (P =.944). However, there was a significant difference between quartiles for %CD63 expression (P =.013) and for CD61/CD14 expression (P =.018), with the lowest quartile demonstrating elevated platelet activation., Conclusions: We found evidence that enhanced platelet activation is associated with reduced plasma volume, but not with plasma catecholamine concentrations. There was no association of platelet concentration with reduced plasma volume. We speculate that elements of the clinical syndrome of preeclampsia coexist as a subclinical phenotype before pregnancy.

The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and "acquired" hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5x normal platelets (10 x 10(8)/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 x 10(8)/mL activated platelets (5x normal); (d) with TF, 10 nM rFVIIa and 3-5x normal nonactivated platelets (6-10 x 10(8)/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.

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.

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.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.

After vascular injury, pericytes may function in blood coagulation events that lead to thrombin formation due to their subendothelial location in the microvasculature. Pericytes from human cerebral cortex microvessels were isolated and characterized, and their ability to express and regulate procoagulant enzyme complexes was determined. Tissue factor was detected on the cell surface of cultured human brain pericytes by immunocytochemistry and was shown to form a functional complex with factor (F) VIIa to effect both FIX and FX activation. Treatment of pericytes with the calcium ionophore A23187 increased the observed tissue factor activity twofold to fivefold, which was shown to be due to an enhancement of cofactor activity and not the release of endogenous antigen stores. Pericytes also provided the appropriate membrane surface required for the assembly of a functional prothrombinase complex, so that in the presence of FVa and FXa, they effected thrombin formation 50 to 100 times faster than any other cell examined to date. In marked contrast to observations in other cell systems, pericyte expression of prothrombinase activity remained unaltered after treatment with A23187. As has been shown for platelets, the membrane receptor on pericytes for FXa assembly into the prothrombinase complex appears to at least partially consist of the FXa receptor effector cell protease receptor-1. These combined data indicate that pericytes can activate and propagate the coagulant response through the extrinsic pathway and that the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation. These observations support the concept that pericytes may play an important role in regulating coagulation events after cerebrovascular injury.

Prior studies using the mutant thrombin, thrombin Quick I, indicate that this protease induces maximum platelet aggregation and intraplatelet [Ca2+] fluxes at agonist concentrations where dissociable, equilibrium platelet binding is undetectable and led to the conclusion that thrombin interaction with its platelet "receptor" is best described kinetically by formation of an enzyme-substrate complex. This conclusion was substantiated further in the present studies by demonstrating that both thrombin Quick I and thrombin mimicked the thrombin receptor agonist peptide in the induction of the platelet activation-dependent events required for functional Prothrombinase assembly and that a rabbit antibody raised against KATNATLDPRSFLLR, a pentadecapeptide which represents amino acids 32-46 in the platelet thrombin receptor/substrate and spans the thrombin cleavage site, inhibited both thrombin- and thrombin Quick I-induced platelet activation responses equivalently. The antipeptide antibody had a more pronounced inhibitory effect on the rate of the thrombin-induced response rather than the magnitude of the response suggesting competition for the cleavage site, consistent with the observation that pretreatment of metabolically-inhibited platelets with thrombin, which was removed by washing, eliminated specific antibody binding due to removal and/or masking of antibody epitopes. Concentrations of the antipeptide antibody that inhibited thrombin- and thrombin Quick I-induced increases in intracellular [Ca2+] flux by as much as 97% did not alter the dissociable equilibrium binding of 125-I-FPR-thrombin to platelets. These combined data indicate that the hydrolytic event initiated by thrombin or thrombin Quick I interaction with the platelet receptor/substrate for thrombin is unrelated to the dissociable equilibrium binding of thrombin to membrane sites described previously by classical receptor-ligand binding analyses.