Total Retention Liquid Chromatography-Mass Spectrometry to Achieve Maximum Protein Sequence Coverage.

Peptide identification by liquid chromatography-mass spectrometry (LC-MS) requires retention and elution of peptides from the LC column. Although medium and hydrophobic peptides are readily retained by the C18 columns that are commonly used in proteomics, short and hydrophilic peptides are not retained nor measured by MS due to their elution in the void volume after sample injection. These nonretained peptides can possess important post-translational modifications, such as glycosylation or phosphorylation. We describe a total retention LC-MS method that employs a reverse phase C18 column and porous graphitic carbon (PGC) column to retain both hydrophobic and hydrophilic peptides for LC-MS analysis. Our setup uses a single valve with a trapping column and two LC pumps run at low microliter/minute flow rates to deliver separate gradients to parallel capillary C18 and PGC columns. Our capillary LC system balances the need for high sensitivity with ease of implementation as compared to other 2D LC systems that use nanocolumns with multiple trapping columns and multiport valves. We demonstrate the utility of the method identifying hydrophilic peptides that went undetected when only a C18 nanocolumn was used. These missed hydrophilic peptides include tripeptides and N-glycosylated species.

Platelets and platelet-derived factor Va confer hemostatic competence in complete factor V deficiency.

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.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation.

Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIß. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIß and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIß in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIß acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

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

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.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3µM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

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 Ibα, and integrins α(IIb) and ß(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.

Platelets: developmental biology, physiology, and translatable platforms for preclinical investigation and drug development.

This paper, developed from the proceedings of the 2007 Platelet Colloquium, considers emerging constructs in platelet biology, preclinical models of thrombosis, and their potential application to the development of platelet-directed pharmacotherapies. Discussed first is the developmental biology of platelets, including megakaryocyte maturation, and the role of apoptotic and growth factors and other proteins in thrombopoiesis. A brief overview of current methods and observations from platelet proteomic analyses is also presented, illustrating the complex interplay of genes, gene expression, protein expression, and protein modification in various atherothrombotic phenotypes. The factor Xa-platelet interface is used as a working model for discussion of anticoagulants as platelet antagonists, highlighting the importance of receptor expression, substrate binding kinetics, platelet subpopulations, and cofactors in thrombosis. Finally, we discuss the use of emerging technologies--such as intravital microscopy and ex vivo perfusion chambers--as translatable platforms for investigating the role of platelets and their pharmacologic inhibition in human health and disease.

Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor: characterization of purified platelet-derived factor V/Va.

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from approximately 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser(692) catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr(402) with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser(692) remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr(1543) rather than Arg(1545) accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.

Carbohydrate moieties on the procofactor factor V, but not the derived cofactor factor Va, regulate its inactivation by activated protein C.

Factor V (FV) is a single-chain plasma protein containing 13-25% carbohydrate by mass. Studies were done to determine if these carbohydrate moieties altered the activated protein C (APC)-catalyzed cleavage and inactivation of both FV and the cofactor which results from its activation by alpha-thrombin, factor Va(IIa) (FVa(IIa)). Treatment of purified FV with N-glycanase and neuraminidase under nonprotein-denaturing conditions removed approximately 20-30% of the carbohydrate from the heavy chain region of the molecule. When glycosidase-treated FV was analyzed in an aPTT (activated partial thromboplastin time)-based APC sensitivity assay, the APC sensitivity ratio (APC-SR) increased from 2.34 to 3.33. In contrast, when glycosidase-treated FV was activated with alpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produced no substantial increase in the APC-SR. Additional functional analyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components indicated that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and were inactivated at identical rates. Analyses of the APC-catalyzed cleavage of glycosidase-treated FV at Arg(306), the initial cleavage site, revealed a 10-fold rate increase when compared to untreated FV. In contrast, and consistent with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-identical rates of APC-catalyzed cleavage at both the Arg(506) and Arg(306)sites. These combined results indicate that N-linked carbohydrate moieties play a substantial role in the APC-catalyzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) cleavage sites of FV and FVa(IIa) are distinct substrates for APC.