Beth A. Bouchard

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.

Human Brain Pericytes Differentially Regulate Expression of Procoagulant Enzyme Complexes Comprising the Extrinsic Pathway of Blood Coagulation

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.

How factor VIIa works in hemophilia.

Summary.  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 5× normal platelets (10 × 108/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 × 108/mL activated platelets (5× normal); (d) with TF, 10 nM rFVIIa and 3–5× normal nonactivated platelets (6–10 × 108/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 in Tumor Progression: A Host Factor That Offers Multiple Potential Targets in the Treatment of Cancer

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. J. Cell. Physiol. 229: 1005–1015, 2014.

No evidence for tissue factor on platelets.

To the editor: Blood coagulation is initiated in vivo by formation of a tissue factor (TF)/factor VIIa (FVIIa) complex.[1][1] Under physiologic conditions, TF is sequestered from blood and coagulation is initiated only after vascular injury. This paradigm has been challenged by studies suggesting

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

Summary.  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.

Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism.

Background: The key source of prothrombin activation in vivo is prothrombinase assembled on the activated platelet surface. Results: Platelet-associated prothrombinase utilizes the prethrombin-2 pathway of prothrombin activation and a concerted enzyme mechanism. Conclusion: Platelet-associated prothrombinase activates prothrombin with a concerted mechanism in which no anticoagulant intermediates are released. Significance: Platelet-associated prothrombinase promotes coagulation by avoiding the release of catalytically active meizothrombin. 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.

Properties of Procoagulant Platelets: Defining and Characterizing the Subpopulation Binding a Functional Prothrombinase

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 Ib&agr;, and integrins &agr;IIb and &bgr;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. Conclusion—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.

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

Summary.  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 Ca2+‐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 [125I]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 α‐granules and not destined for lysosomal degradation.

Interactions Between Platelets and the Coagulation System

These events amplify the initating stimulus and lead to explosive thrombin generation and fibrinformation. Platelets can also sustain the procoagulant response by protecting the bound coagulation enzyme complexes from inactivation/inhibition. This overall process provides for rapid and localized clot formation Thrombin generation at the surface of platelets adhered to and activated at sites of vascular injury is central to formation of an insoluble fibrin clot and localized cessation of bleeding. Those platelets that actively regulate thrombin formation can be defined by: (1) a discrete population of young platelets expressing an increased density of adhesion molecules; (2) expression of specific, coagulation protein receptors; and (3) release of a number of hemostatically-active components including a unique and highly procoagulant factor Va molecule.