Subia Tasneem

Mice with deleted multimerin 1 and α-synuclein genes have impaired platelet adhesion and impaired thrombus formation that is corrected by multimerin 1☆

BACKGROUND Multimerin 1 is a stored platelet and endothelial cell adhesive protein that shows significant conservation. In vitro, multimerin 1 supports platelet adhesion and it also binds to collagen and enhances von Willebrand factor-dependent platelet adhesion to collagen. As selective, multimerin 1 deficient mice have not been generated, we investigated multimerin 1 effects on platelet adhesion using a subpopulation of C57BL/6J mice with tandem deletion of the genes for multimerin 1 and alpha-synuclein, a protein that inhibits alpha-granule release in vitro. We postulated that multimerin 1/alpha-synuclein deficient mice might show impaired platelet adhesive function from multimerin 1 deficiency and increased alpha-granule release from alpha-synuclein deficiency. METHODS Platelet function was assessed by intravital microscopy, after ferric chloride injury, using untreated and human multimerin 1-transfused multimerin 1/alpha-synuclein deficient mice, and by in vitro assays of adhesion, aggregation and thrombin-induced P-selectin release. RESULTS Multimerin 1/alpha-synuclein deficient mice showed impaired platelet adhesion and their defective thrombus formation at sites of vessel injury improved with multimerin 1 transfusion. Although multimerin 1/alpha-synuclein deficient platelets showed increased P-selectin release at low thrombin concentrations, they also showed impaired adhesion to collagen, and attenuated aggregation with thrombin, that improved with added multimerin 1. CONCLUSIONS Our data suggest that multimerin 1 supports platelet adhesive functions and thrombus formation, which will be important to verify by generating and testing selective multimerin 1 deficient mice.

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.

Simultaneous measurement of adenosine triphosphate release and aggregation potentiates human platelet aggregation responses for some subjects, including persons with Quebec platelet disorder

Platelet aggregometry and dense granule adenosine triphosphate (ATP) release assays are helpful to diagnose platelet disorders. Some laboratories simultaneously measure aggregation and ATP release using Chronolume® a commercial reagent containing D-luciferin, firefly luciferase and magnesium. Chronolume® can potentiate sub-maximal aggregation responses, normalising canine platelet disorder findings. We investigated if Chronolume® potentiates human platelet aggregation responses after observing discrepancies suspicious of potentiation. Among patients simultaneously tested by light transmission aggregometry (LTA) on two instruments, 18/43 (42%), including 14/24 (58%) with platelet disorders, showed full secondary aggregation with one or more agonists only in tests with Chronolume®. As subjects with Quebec platelet disorder (QPD) did not show the expected absent secondary aggregation responses to epinephrine in tests with Chronolume®, the reason for the discrepancy was investigated using samples from 10 QPD subjects. Like sub-threshold ADP (0.75 μM), Chronolume® significantly increased QPD LTA responses to epinephrine (p<0.0001) and it increased both initial and secondary aggregation responses, leading to dense granule release. This potentiation was not restricted to QPD and it was mimicked adding 1-2 mM magnesium, but not D-luciferin or firefly luciferase, to LTA assays. Chronolume® potentiated the ADP aggregation responses of QPD subjects with a reduced response. Furthermore, it increased whole blood aggregation responses of healthy control samples to multiple agonists, tested at concentrations used for the diagnosis of platelet disorders (p values <0.05). Laboratories should be aware that measuring ATP release with Chronolume® can potentiate LTA and whole blood aggregation responses, which alters findings for some human platelet disorders, including QPD.

Platelet adhesion to multimerin 1 in vitro: influences of platelet membrane receptors, von Willebrand factor and shear

Summary.  Background: Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein, stored in platelets and endothelium, that when released, binds to activated platelets, endothelial cells and the extracellular matrix. Objectives: The goals of our study were to determine if (i) MMRN1 supports adhesion of resting and/or activated platelets under conditions of blood flow, and (ii) if MMRN1 enhances platelet adhesion to types I and III collagen. Patients/methods: Platelet adhesion was evaluated using protein‐coated microcapillaries, with or without added adhesive proteins and receptor antibodies. Platelets from healthy controls, Glanzmann thrombasthenia (GT) and severe von Willebrand factor (VWF)‐deficient donors were tested. Results: MMRN1 supported the adhesion of activated, but not resting, washed platelets over a wide range of shear rates. At low shear (150 s−1), this adhesion was supported by integrins αvβ3 and glycoprotein (GP) Ibα but it did not require integrins αIIbβ3 or VWF. At high shear (1500 s−1), adhesion to MMRN1 was supported by β3 integrin‐independent mechanisms, involving GPIbα and VWF, that did not require platelet activation when VWF was perfused over MMRN1 prior to platelets. MMRN1 bound to types I and III collagen, independent of VWF, however, its enhancing effects on platelet adhesion to collagen at high shear were VWF dependent. Conclusions: MMRN1 supports platelet adhesion by VWF‐dependent and ‐independent mechanisms that vary by flow rate. Additionally, MMRN1 binds to, and enhances, platelet adhesion to collagen. These findings suggest that MMRN1 could function as an adhesive ligand that promotes platelet adhesion at sites of vascular injury.

An acquired factor V inhibitor associated with defective factor V function, storage and binding to multimerin 1.

pathway inhibitor: regulation of its inhibitory activity by phospholipid surfaces. Haemostasis 1996; 26(Suppl. 4): 89–97. 6 Hackeng TM, Sere KM, Tans G, Rosing J. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci U S A 2006; 103: 3106–11. 7 Dahm AE, Andersen TO, Rosendaal F, Sandset PM. A novel anticoagulant activity assay of tissue factor pathway inhibitor I (TFPI). J Thromb Haemost 2005; 3: 651–8 . 8 Koster T, Rosendaal FR, Briet E, van der Meer FJ, Colly LP, Trienekens PH, Poort SR, Reitsma PH, Vandenbroucke JP. Protein C deficiency in a controlled series of unselected outpatients: an infrequent but clear risk factor for venous thrombosis (Leiden Thrombophilia Study). Blood 1995; 85: 2756–61. 9 Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503–6. 10 Vossen CY, Conard J, Fontcuberta J, Makris M, van der Meer FJ, Pabinger I, Palareti G, Preston FE, Scharrer I, Souto JC, Svensson P, Walker ID, Rosendaal FR. Familial thrombophilia and lifetime risk of venous thrombosis. J Thromb Haemost 2004; 2: 1526–32.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.

The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding

Summary Multimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbα binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

Thrombopoietin levels in Quebec platelet disorder-Implications for the mechanism of thrombocytopenia

Thrombopoietin (TPO) is the primary regulator of platelet production.1,2 TPO levels in plasma are increased in disorders that impair megakaryocyte and platelet production, such as aplastic anemia, and are normal in thrombocytopenic disorders that do not reduce the megakaryocyte mass, including those that accelerate platelet clearance.1,2 We postulated that a detailed analysis of TPO levels in Quebec platelet disorder (QPD) might provide insights into why platelet counts are reduced in this inherited bleeding disorder3 which is caused by a duplication mutation of PLAU, the urokinase plasminogen activator (uPA) gene.4,5 QPD markedly and selectively increases the production of normal PLAU transcripts by the disease chromosome in megakaryocytes but not in leukocytes through unknown mechanisms,6 and this increases megakaryocyte and platelet uPA levels by more than 100fold.7,8 As QPD megakaryocytes and platelets sequester uPA in αgranules,8 the disorder results in a unique, platelet activationdependent, gainoffunction defect in fibrinolysis, without systemic fibrinolysis.9 In QPD, platelet morphology (by light and electron microscopy), size,3 and dense granule release are normal, and secondary platelet aggregation is absent with epinephrine, with or without reduced maximal aggregation with collagen, adenosine diphosphate, and/or thromboxane analog U46619.10 Many persons with QPD have thrombocytopenia as platelet counts in this disorder are typically about 50% lower than the platelet counts of unaffected relatives (reported range: ~120245 × 109/L11). The reduction in platelet counts in QPD is clinically significant as lower platelet counts are associated with wound healing problems.11 To gain insights into the reduced platelet counts in QPD, we evaluated TPO levels and platelet counts with the approval of the Hamilton Integrated Research Ethics Board and the Research Ethics Board of Centre Hospitalier Universitaire SainteJustine and written informed consent of blood donors. Plateletpoor plasma for TPO analyses was prepared using EDTAanticoagulated blood (collected using vacutainers) from QPD (n = 7 representative individuals with the QPD genetic mutation who had often donated samples for research4-11) and general population control participants (n = 12). Plasmas were tested using the Quantikine® ELISA Human TPO Immunoassay (R&D Systems, Minneapolis, MN, USA), as recommended, with TPO values below the lowest assay standard (31.3 pg/mL) reported as <31.3 pg/mL. Although QPD participants had lower platelet counts than control participants (respective medians [ranges]: 143 [116198] × 109/L vs 223 [165297] × 109/L; P < .01 based on MannWhitney test), all QPD and all but one of the control participants (TPO level: 59 pg/mL) had plasma TPO levels <31.3 pg/mL. Accordingly, all participants had plasma TPO levels within the range of expected results for healthy volunteers, which are typically <31.3 pg/mL but can be as high as 196 pg/ mL, according to the ELISA manufacturer. The reasons for thrombocytopenia in QPD could be complex. TPO cleavage by uPA increases its activity, whereas TPO cleavage by plasmin has the opposite effect.12 As TPO levels in QPD plasma were <31.3 pg/mL, we were unable to investigate whether QPD causes increased TPO proteolysis. The sequestration of uPA in QPD αgranules (which prevents systemic fibrinolysis)9 might limit TPO proteolysis by uPA and plasmin in QPD. It is also possible that the increased uPA in QPD megakaryocytes is insufficient to increase TPO effects on megakaryocytes given that there are normal expansion and differentiation of QPD peripheral blood CD34+ stem cells into megakaryocytes in serumfree cultures with added TPO.8 While the cause of QPD thrombocytopenia has not been resolved, it is possible that increased plasmin generation is part of the pathogenesis as treatment with fibrinolytic inhibitor drugs, such as tranexamic acid, corrects both the bleeding and wound healing problems of QPD (CPMH and GER, unpublished observations). Most individuals with QPD require only short courses with fibrinolytic inhibitor drugs (eg, 210 days) for preventing and treating bleeding (eg, for dental procedures, surgery, complicated childbirth, joint bleeds, and traumarelated bleeding). The possibility that fibrinolytic inhibitor therapy might improve QPD thrombocytopenia led us to examine the platelet count records for 31 individuals with QPD, which included the individuals who donated samples for TPO analysis (n = 10 females, n = 21 males; ages: 477 years). The recorded platelet counts for persons with QPD ranged from 92448 × 109/L, after excluding the person whose platelets fell to 19 × 109/L during neutropenia unrelated to QPD. Most persons with QPD had fairly stable platelet counts, and while 29% (9/31) had platelet counts below <150 × 109/L on the majority of tests, 48% (15/31) had thrombocytopenia documented at least once. A few persons with QPD had ~2.9fold increases in their platelet counts while on fibrinolytic inhibitor therapy after surgery, and these individuals had the highest recorded QPD platelet counts. As platelet counts normally increase in the postoperative period, more data are needed on QPD platelet counts before, during, and after fibrinolytic inhibitor therapy to determine whether this treatment corrects QPD thrombocytopenia.