Genetic loci associated with platelet traits and platelet disorders.

Genetic investigations have led to important advances in our knowledge of genes, proteins, and microRNA that influence circulating platelet counts, platelet size, and function. The application of genome-wide association studies (GWAS) to platelet traits has identified multiple loci with a significant association to platelet number, size, and function in aggregation and granule secretion assays. Moreover, the genes altered by disease-causing mutations have now been identified for several platelet disorders, including X-linked recessive, autosomal dominant, and autosomal recessive platelet disorders. Some mutations that cause inherited platelet disorders involve genes that GWAS have associated to platelet traits. Although disease-causing mutations in many rare and syndromic causes of platelet disorders have now been characterized, the genetic mutations that cause common inherited platelet disorders, and impair platelet aggregation and granule secretion, are largely unknown. This review summarizes current knowledge on the genetic loci that influence platelet traits, including the genes with well-characterized mutations in certain inherited platelet disorders.

Adjuvanted trivalent influenza vaccine versus quadrivalent inactivated influenza vaccine in Hutterite Children: A randomized clinical trial.

BACKGROUND: Children play an important role in the transmission of influenza. The best choice of vaccine to achieve both direct and indirect protection is uncertain. The objective of the study was to test whether vaccinating children with MF59 adjuvanted trivalent influenza vaccine (aTIV) can reduce influenza in children and their extended households compared to inactivated quadrivalent vaccine (QIV). METHODS: We conducted a cluster randomized trial in 42 Hutterite colonies in Alberta and Saskatchewan. Colonies were randomized such that children were assigned in a blinded manner to receive aTIV (0.25 ml of pediatric aTIV for ages 6 months to < 36 months or 0.5 ml for ages ≥ 36 months to 6 years) or 0.5 ml of QIV. Participants included 424 children aged 6 months to 6 years who received the study vaccine and 1246 family cluster members who did not receive the study vaccine. The primary outcome was confirmed influenza A and B infection using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay. An intent to treat analysis was used. Data were collected from January 2017 to June 2019. RESULTS: The mean percentage of children who received study vaccine was 62% for aTIV colonies and 74% for QIV colonies. There were 66 (3.4%) with RT-PCR confirmed influenza A and B in the aTIV colonies (children and family clusters) versus 93 (4.4%) in the QIV colonies, hazard ratio (HR) 0.78 (95 %CI 0.36-1.71). Of these, 48 (2.5%) in the aTIV colonies and 76 (3.6%) in the QIV colonies had influenza A, HR 0.69, (95 %CI 0.29-1.66) while 18 (0.9%) and 17 (0.8%) in the aTIV versus QIV colonies respectively had influenza B, HR 1.22, (95 %CI 0.20-7.41). In children who received study vaccine, there were 5 Influenza A infections in the aTIV colonies (1.1%) compared to 30 (5.8%) in the QIV colonies, relative efficacy of 80%, HR 0.20, (95 %CI 0.06-0.66). Adverse events were significantly more common among children who received aTIV. No serious vaccine adverse events were reported. CONCLUSION: Vaccinating children with aTIV compared to QIV resulted in similar community RT-PCR confirmed influenza illness and led to significant protection against influenza A in children.

Multimerin 1 binds factor V and activated factor V with high affinity and inhibits thrombin generation.

Multimerin 1 (MMRN1) is a polymeric, factor V (FV) binding protein that is stored in platelet and endothelial cell secretion granules but is undetectable in normal plasma. In human platelet alpha-granules, FV is stored complexed to MMRN1, predominantly by noncovalent binding interactions. The FV binding site for MMRN1 is located in the light chain, where it overlaps the C1 and C2 domain membrane binding sites essential for activated FV (FVa) procoagulant function. Surface plasmon resonance (SPR), circular dichroism (CD) and thrombin generation assays were used to study the binding of FV and FVa to MMRN1, and the functional consequences. FV and FVa bound MMRN1 with high affinities (K(D): 2 and 7 nM, respectively). FV dissociated more slowly from MMRN1 than FVa in SPR experiments, and CD analyses suggested greater conformational changes in mixtures of FV and MMRN1 than in mixtures of FVa and MMRN1. SPR analyses indicated that soluble phosphatidylserine (1,2-Dicaproylsn-glycero-3-phospho-L-serine) competitively inhibited both FV-MMRN1 and FVa-MMRN1 binding. Furthermore, exogenous MMRN1 delayed and reduced thrombin generation by plasma and platelets, and it reduced thrombin generation by preformed FVa. Exogenous MMRN1 also delayed FV activation, triggered by adding tissue factor to plasma, or by adding purified thrombin or factor Xa to purified FV. The high affinity binding of FV to MMRN1 may facilitate the costorage of the two proteins in platelet alpha-granules. As a consequence, MMRN1 release during platelet activation may limit platelet dependent thrombin generation in vivo.

Location of the multimerin 1 binding site in coagulation factor V: an update.

Activated coagulation factor V (FVa) is an important cofactor that accelerates thrombin production. In human blood, 25% of the factor V (FV) is stored in platelets, complexed to the polymeric, FV binding protein multimerin 1 (MMRN1). The light chain of FV is required for MMRN1 binding, and its C2 domain contains a MMRN1 binding site that overlaps phospholipid binding residues essential for FVa procoagulant function. The homologous structures and roles of the FVa light chain C1 and C2 domains led us to investigate if the C1 domain also contains a MMRN1 binding site. The MMRN1 binding properties of FV constructs were tested by modified enzyme-linked immunoassays, before and after thrombin activation. The constructs tested included the combined C1 and C2 domain deleted FV, and B-domain deleted forms of FV containing C1 domain point mutations or combined C1 and C2 domain phospholipid binding site mutations. The MMRN1 binding site in FV/FVa was mapped to a large region that included the C1 domain phospholipid binding residues Y1956 and L1957. The FV construct with combined C1 and C2 domain phospholipid binding site mutations had no MMRN1 binding, highlighting the critical role of the FV C1 and C2 domain phospholipid binding residues in MMRN1 binding. Our data update the information on the structural features of FV and FVa important for MMRN1 binding, and suggest that the extended MMRN1 binding site in the C1 and C2 domains is important for the storage of FV-MMRN1 complexes in platelets.

Defective membrane interactions of familial Parkinson's disease mutant A30P alpha-synuclein.

alpha-Synuclein (alpha-Syn) is an abundant presynaptic protein of unknown function, which has been implicated in the pathogenesis of Parkinson's disease. Alpha-Syn has been suggested to play a role in lipid transport and synaptogenesis, and growing evidence suggests that alpha-Syn interactions with cellular membranes are physiologically important. In the current study, we demonstrate that the familial Parkinson's disease-linked A30P mutant alpha-Syn is defective in binding to phospholipid vesicles in vitro as determined by vesicle ultracentrifugation, circular dichroism spectroscopy, and low-angle X-ray diffraction. Interestingly, our data also suggest that alpha-Syn may bind to the lipid vesicles as a dimer, which suggest that this species could be a physiologically relevant and functional entity. In contrast, the naturally occurring murine A53T substitution, which is also linked to Parkinson's disease, displayed a normal membrane-binding activity that was comparable to wild-type alpha-Syn. A double mutant A53T/A30P alpha-Syn showed defective membrane binding similar to the A30P protein, indicating that the proline mutation is dominant in terms of impairing the membrane-binding activity. With these observations, we suggest that the A53T and A30P mutants may have different physiological consequences in vivo and could possibly contribute to early onset Parkinson's disease via unique mechanisms.

Endocytosis and storage of plasma factor V by human megakaryocytes.

Factor V is an essential coagulation cofactor that circulates in plasma and platelet alpha-granules where it is stored complexed to multimerin I (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN I complexes, we studied factorV in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factor V, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte alpha-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into alpha-granules and generate factorV-MMRN I complexes from endogenously and exogenously synthesized proteins.

Human platelets contain forms of factor V in disulfide-linkage with multimerin.

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Elastic properties of polyunsaturated phosphatidylethanolamines influence rhodopsin function.

Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (M(II)), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether M(II)-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18 : 0-22 : 6(n - 3)PE, 18 : 0-22 : 5(n)- 6PE and the model lipid 18 : 1(n - 9)-18 : 1,(n- 9)PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 angstroms. In membranes, all three PEs generate high negative curvature elastic stress that shifts the equilibrium of MI(I)/M(II) photointermediates of rhodopsin towards M(II) formation.

Spontaneous curvature of phosphatidic acid and lysophosphatidic acid.

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R(0p)) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R(0p) values of PA and LPA were -46 A and +20 A, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca(2+) did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R(0p) of -1.3 x 10(2) A). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.

Curvature and bending constants for phosphatidylserine-containing membranes.

Phosphatidylserine (PS), an anionic phospholipid of significant biological relevance, forms a multilamellar phase in water with net negative surface charge at pH 7.0. In this study we mixed dioleoylPS (DOPS) with reverse hexagonal (H(II))-forming phosphatidylethanolamine (DOPE), and used x-ray diffraction and osmotic stress to quantify its spontaneous curvature (1/R(0p)) and bending modulus (K(cp)). The mixtures were stable H(II) phases from 5 to 30 mol% PS, providing 16 wt% tetradecane (td) was also added to relieve chain-packing stress. The fully hydrated lattice dimension increased with DOPS concentration. Analysis of structural changes gave an apparent R(0p) for DOPS of +144 A; opposite in sign and relatively flat compared to DOPE (-30 A). Osmotic stress of the H(II) phases did not detect a significantly different bending modulus (K(cp)) for DOPS as compared to DOPE. At pH < or = 4.0, DOPS (with no td) adopted the H(II) phase on its own, in agreement with previous results, suggesting a reversal in curvature upon protonation of the serine headgroup. In contrast, when td was present, DOPS/td formed a lamellar phase of limited swelling whose dimension increased with pH. DOPS/DOPE/td mixtures formed H(II) phases whose dimension increased both with pH and with DOPS content. With tetradecane, estimates put 1/R(0p) for DOPS at pH 2.1 at zero. Tetradecane apparently affects the degree of dissociation of DOPS at low pH.

The effects of acyl chain length and saturation of diacylglycerols and phosphatidylcholines on membrane monolayer curvature.

The second messenger, diacylglycerol (DAG), introduces negative curvature in phospholipid monolayers and strongly induces the lamellar (L(alpha)) to reverse hexagonal (H(II)) phase transition. The chain lengths and degree of unsaturation of symmetric DAGs influence this effect. Within dioleoylphosphatidylcholine (DOPC) monolayers, the apparent spontaneous radius of curvature (R(0)) of the short, saturated dicaprylglycerol (C10-DCG) itself was determined to be -13.3 A, compared with an R(0) value of -10.1 A for the long, di-monounsaturated dioleoylglycerol (C18-DOG). Such increased length and unsaturation of the DAG acyl chains produces this small change. Di-saturated phosphatidylcholines (PCs) with equal length chains (from C10-C18) with 25 mol % DOG do not form the H(II) phase, even under the unstressed conditions of excess water and alkane. Di-unsaturated PCs with equal chain length (from C14-C18) with 25 mol % DOG do form the H(II) phase. Asymmetric chained PCs (position 1 saturated with varying lengths, position 2 differentially unsaturated with varying lengths) all form the H(II) phase in the presence of 25 mol % DOG. As a general rule for PCs, their unsaturation is critical for the induction of the H(II) phase by DOG. The degree of curvature stress induced by the second messenger DOG in membranes, and any protein that might be affected by it, would appear to depend on chain unsaturation of neighboring PCs.

Identification of the MMRN1 binding region within the C2 domain of human factor V.

In platelets, coagulation cofactor V is stored in complex with multimerin 1 in alpha-granules for activation-induced release during clot formation. The molecular nature of multimerin 1 factor V binding has not been determined, although multimerin 1 is known to interact with the factor V light chain. We investigated the region in factor V important for multimerin 1 binding using modified enzyme-linked immunoassays and recombinant factor V constructs. Factor V constructs lacking the C2 region or entire light chain had impaired and absent multimerin 1 binding, respectively, whereas the B domain deleted construct had modestly reduced binding. Analyses of point mutated constructs indicated that the multimerin 1 binding site in the C2 domain of factor V partially overlaps the phosphatidylserine binding site and that the factor V B domain enhances multimerin 1 binding. Multimerin 1 did not inhibit factor V phosphatidylserine binding, and it bound to phosphatidylserine independently of factor V. There was a reduction in factor V in complex with multimerin 1 after activation, and thrombin cleavage significantly reduced factor V binding to multimerin 1. In molar excess, multimerin 1 minimally reduced factor V procoagulant activity in prothrombinase assays and only if it was added before factor V activation. The dissociation of factor V-multimerin 1 complexes following factor V activation suggests a role for multimerin 1 in delivering and localizing factor V onto platelets prior to prothrombinase assembly.