Human platelet antigen polymorphisms (HPA‐1, ‐2, ‐3, ‐4, ‐5 and ‐15) in major ethnic groups of Pakistan

Objectives: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA‐typed platelet donors in a given community. Background: However, the pattern of HPA in Pakistani population is not known. Aim: The aim of present study was to determine the gene frequencies of HPA (HPA‐1 to ‐5 and ‐15) in individuals belonging to major ethnic groups and castes of Pakistani population. Materials and Methods: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction‐sequence specific primers with detection on polyacrylamide electrophoresis. Results: The gene frequencies of the ‘a’ and ‘b’ alleles of HPA‐1 to ‐5 and ‐15 in Pakistanis were as follows: HPA‐1a/b, 0·885/0·115; HPA‐2a/b, 0·92/0·08; HPA‐3a/b, 0·69/0·31; HPA‐4a/b, 1/0; HPA‐5a/b, 0·9/0·1; HPA‐15a/b, 0·59/0·41. Except for significant difference regarding gene frequency of HPA‐3 between Pathans and Sindhis, there was no significant difference of HPA‐1 to ‐5 and ‐15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1–5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti‐HPA‐1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8–12 of 1000 in case of anti‐HPA‐5b. Homozygosity of HPA‐1b, ‐2b and ‐5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA‐3b and ‐15b was 11 and 18%. Conclusions: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.     

Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP‐induced platelet activation

Chang H, Yanachkov IB, Dix EJ, Li YF, Barnard MR, Wright GE, Michelson AD, Frelinger AL 3rd. Modified diadenosine tetraphosphates with dual specificity for P2Y₁ and P2Y₁₂ are potent antagonists of ADP‐induced platelet activation. J Thromb Haemost 2012; 10: 2573–80. Summary. Background: Diadenosine 5′,5′′′‐P¹,P⁴‐tetraphosphate (Ap₄A), a natural compound stored in platelet dense granules, inhibits ADP‐induced platelet aggregation. Ap₄A inhibits the platelet ADP receptors P2Y₁ and P2Y₁₂, is a partial agonist of P2Y₁₂, and is a full agonist of the platelet ATP‐gated ion channel P2X1. Modification of the Ap₄A tetraphosphate backbone enhances inhibition of ADP‐induced platelet aggregation. However, the effects of these Ap₄A analogs on human platelet P2Y₁, P2Y₁₂ and P2X1 are unclear. Objective: To determine the agonist and antagonist activities of diadenosine tetraphosphate analogs towards P2Y₁, P2Y₁₂, and P2X1. Methods: We synthesized the following Ap₄A analogs: P¹,P⁴‐dithiotetraphosphate; P²,P³‐chloromethylenetetraphosphate; P¹‐thio‐P²,P³‐chloromethylenetetraphosphate; and P¹,P⁴‐dithio‐P²,P³‐chloromethylenetetraphosphate. We then measured the effects of these analogs on: (i) ADP‐induced platelet aggregation; (ii) P2Y₁‐mediated changes in cytosolic Ca²⁺; (iii) P2Y₁₂‐mediated changes in vasodilator‐stimulated phosphoprotein phosphorylation; and (iv) P2X1‐mediated entry of extracellular Ca²⁺.Results: Ap₄A analogs with modifications in the phosphate backbone inhibited both P2Y₁ and P2Y₁₂, and showed no agonist activity towards these receptors. The dithio modification increased inhibition of P2Y₁, P2Y₁₂, and platelet aggregation, whereas the chloromethylene modification increased inhibition of P2Y₁₂ and platelet aggregation, but decreased P2Y₁ inhibition. Combining the dithio and chloromethylene modifications increased P2Y₁ and P2Y₁₂ inhibition. As compared with Ap₄A, each modification decreased agonist activity towards P2X1, and the dual modification completely eliminated P2X1 agonist activity. Conclusions: As compared with Ap₄A, tetraphosphate backbone analogs of Ap₄A have diminished activity towards P2X1 but inhibit both P2Y₁ and P2Y₁₂ and, with greater potency, inhibit ADP‐induced platelet aggregation. Thus, diadenosine tetraphosphate analogs with dual receptor selectivity may have potential as antiplatelet drugs.     

A platelet tetraspanin superfamily member,CD151, is required for regulation of thrombus growth and stability in vivo

Summary. Background: This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. Methods and Results: Using intravital microscopy and ferric chloride (FeCl₃) injury of mesenteric arterioles, we found that thrombi formed in CD151^+/? and CD151^?/? mice were smaller and less stable, than those formed in CD151^+/+ mice, with a tendency for embolization. Similarly, in Folt’s FeCl₃?induced carotid injury model, both CD151^+/? and CD151^?/? mice showed more prolonged times to 95% vessel occlusion than CD151^+/+ mice. In addition, laser‐induced injury of cremaster muscle arterioles showed that thrombi formed in CD151^+/? and CD151^?/? mice were smaller and less stable than those formed in CD151^+/+ mice. Following platelet depletion/reconstitution with ex vivo‐labeled donor platelets, platelet‐depleted CD151^+/+ mice that received reconstitution with CD151^?/? platelets had smaller thrombi that were unstable and embolized. In contrast, platelet‐depleted CD151^?/? mice that received reconstitution with CD151^+/+ platelets had normal thrombi that were stable. Conclusions: These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.     

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC‐2

Summary. Background: The adapter proteins SLP‐76 and LAT have been shown to play critical roles in the activation of PLCγ2 in platelets downstream of GPVI/FcRγ and the C‐type lectin receptor CLEC‐2. SLP‐76 is constitutively associated with the adapter Gads in platelets, which also binds to tyrosine phosphorylated LAT, thereby providing a potential pathway of regulation of SLP‐76. Objective: In the present study, we have compared the role of Gads alongside that of LAT following activation of the major platelet glycoprotein receptors using mice deficient in the two adapter proteins. Results: Gads was found to be required for the efficient onset of aggregation and secretion in response to submaximal stimulation of GPVI and CLEC‐2, but to be dispensable for activation following stronger stimulation of the two receptors. Gads was also dispensable for spreading induced through integrin α_IIbβ₃ or the GPIb–IX–V complex. Further, Gads plays a negligible role in aggregate formation on collagen at an arteriolar rate of shear. In stark contrast, platelets deficient in the adapter LAT exhibit a marked decrease in aggregation and secretion following activation of GPVI and CLEC‐2, and are unable to form stable aggregates on collagen at arteriolar shear. Conclusions: The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP‐76 in response to weak activation of GPVI and CLEC‐2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads‐independent pathway of platelet activation downstream of LAT.     

Aspirin and the in vitro linear relationship between thromboxane A2‐mediated platelet aggregation and platelet production of thromboxane A2

Summary. Background: Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A₂ formation is non‐linear and TXA₂ generation must be inhibited by at least 95% to inhibit TXA₂‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA₂ generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA₂ production in response to arachidonic acid (0.03–1 mmol L^?1), collagen (0.1–30 μg mL^?1), epinephrine (0.001–100 μmol L^?1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30 μmol L^?1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA₂ production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC₅₀ values that did not differ. A linear relationship existed between aggregation and TXA₂ production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R² 0.92; n = 224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA₂ generation and TXA₂‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

Progenitor‐derived endothelial cell response,platelet reactivity and haemocompatibility parameters indicate the potential of NaOH‐treated polycaprolactone for vascular tissue engineering

The haemocompatibility of NaOH‐treated poly(ε‐caprolactone) (PCL) has been evaluated in vitro by analysing several parameters, including plasma recalcification time, whole blood clotting time and platelet adhesion/activation. NaOH‐treated PCL films showed a significant decrease in the clot formation speed and a reduced number of adhered platelets, which mainly exhibited non‐activated morphologies. Furthermore, mature endothelial cells derived from peripheral endothelial progenitor cells were cultured on the polymer to investigate the effects of the endothelial lining on polymer haemocompatibility. Interestingly, cells cultured on NaOH‐treated PCL films showed a significant stimulation of NO production. Although further research is required, NaOH treatment could be an interesting and simple strategy to modify PCL‐based materials in order to enhance endothelial NO production, where compromised, and provide a better interaction of the scaffold with the blood components. In conclusion, these results reinforce the use of NaOH‐treated PCL as a haemocompatible polymer for vascular tissue‐engineering applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of Hic‐5 as a novel regulatory factor for integrin αIIbβ3 activation and platelet aggregation in mice

Summary. Background: Integrin αIIbβ3 plays key roles in platelet aggregation and subsequent thrombus formation. Hydrogen peroxide‐inducible clone‐5 (Hic‐5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic strand. Objectives: Hic‐5 function in αIIbβ3 activation and subsequent platelet aggregation remains unknown. To address this question, platelets from Hic‐5^?/? mice were analyzed. Methods and Results: Hic‐5^?/? mice displayed a significant hemostatic defect and resistance to thromboembolism, which were explained in part by weaker thrombin‐induced aggregation in Hic‐5^?/? platelets. Mechanistically, Hic‐5^?/? platelets showed limited activation of αIIbβ3 upon thrombin treatment. Morphological alteration in Hic‐5^?/? platelets after thrombin stimulation on fibrinogen plates was also limited. As a direct consequence, the quantity of actin co‐immunoprecipitating with the activated αIIbβ3 was smaller in Hic‐5^?/? platelets than in wild‐type platelets. Conclusion: We identified Hic‐5 as a novel and specific regulatory factor for thrombin‐induced αIIbβ3 activation and subsequent platelet aggregation in mice.     

Clinical management,ethics and informed consent related to multi‐gene panel‐based high throughput sequencing testing for platelet disorders: Communication from the SSC of the ISTH

Molecular diagnostics of inherited platelet disorders (IPD) has been revolutionized by the implementation of high‐throughput sequencing (HTS) approaches. A conclusive diagnosis using HTS tests can be obtained quickly and cost‐effectively in many, but not all patients. The expanding use of HTS tests has raised concerns regarding complex variant interpretation and the ethical implications of detecting unsolicited findings such as variants in IPD genes RUNX1, ETV6, and ANKRD26, which are associated with increased leukemic risk. This guidance document has been developed and written by a multidisciplinary team of researchers and clinicians, with expertise in hematology, clinical and molecular genetics, and bioethics, alongside a RUNX1 patient advocacy representative. We recommend that for clinical diagnostics, HTS for IPD should use a multigene panel of curated diagnostic‐grade genes. Critically, we advise that an HTS test for clinical diagnostics should only be ordered by a clinical expert that is: (a) fully aware of the complexity of genotype‐phenotype correlations for IPD; (b) able to discuss these complexities with a patient and family members before the test is initiated; and (c) able to interpret and appropriately communicate the results of a HTS diagnostic report, including the implication of variants of uncertain clinical significance. Each patient should know what an HTS test could mean for his or her clinical management before initiating a test. We hereby propose an exemplified informed consent document that includes information on these ethical concerns and can be used by the community for implementation of HTS of IPD in a clinical diagnostic setting. This paper does not include recommendations for HTS of IPD in a research setting.     

Comparison of perineural platelet‐rich plasma and dextrose injections for moderate carpal tunnel syndrome: A prospective randomized,single‐blind,head‐to‐head comparative trial

Recent studies demonstrated the utility of perineural injection with platelet‐rich plasma (PRP) and 5% dextrose (D5W) as novel strategies for treatment of carpal tunnel syndrome (CTS). The present study comprised a prospective, randomized, single‐blind, head‐to head comparative trial to compare the 6‐month outcome of perineural injection with PRP or D5W in patients with moderate CTS. Fifty‐two patients with unilateral moderate CTS were enrolled and randomized into two groups: The PRP group received a single 3‐cc perineural injection of PRP under ultrasound guidance, and dextrose group received a single 3‐cc perineural injection of D5W under ultrasound guidance. The Boston Carpal Tunnel Syndrome Questionnaire score was used as the primary outcome. Secondary outcomes included cross‐sectional area (CSA) of the median nerve and electrophysiological assessments. Evaluations were performed at baseline and at 1, 3, and 6 months postinjection. All patients (26 patients per group) completed the study. Compared with the dextrose group, the PRP group demonstrated significant reductions in Boston Carpal Tunnel Syndrome Questionnaire function at 3 months (p = .044), distal motor latency at 6 months (p = .028), and CSA at 3 and 6 months (p = .010 and.018, respectively). A single perineural injection of PRP reduced the CSA of the median nerve more effectively than injection of D5W at 3 and 6 months postinjection for patients with moderate CTS.     

Serum paraoxonase‐I activity is unaffected by short‐term administration of simvastatin,bezafibrate, and their combination in type 2 diabetes mellitus

Background The high‐density lipoprotein (HDL)‐associated anti‐oxidative and anti‐inflammatory enzyme, paraoxonase‐I, has been found previously to be lower in type 2 diabetes mellitus. We studied whether statin and fibrate treatment, alone and in combination, affect serum paraoxonase‐I activity in conjunction with changes in HDL cholesterol in diabetic patients. Subjects and methods A placebo‐controlled crossover study was carried out in 14 type 2 diabetic patients to test the effect of 8 weeks of active treatment with simvastatin (40 mg daily), bezafibrate (400 mg daily), and their combination on serum paraoxonase‐I activity, measured as its activity towards arylesterase and paraoxon. Serum paraoxonase‐I activity was also compared between these diabetic patients and 49 non‐diabetic control subjects. Results Serum arylesterase activity was lower in type 2 diabetic patients compared to control subjects (P < 0·001), but the difference in paraoxonase activity was not significant (P = 0·22). Neither arylesterase (P = 0·24) nor paraoxonase activity (P = 0·37) was increased in response to treatment, despite higher HDL cholesterol and apolipoprotein A‐I during combination therapy (P < 0·05 for both). Conclusion Short‐term administration of simvastatin and bezafibrate, even when combined, is ineffective in raising serum paraoxonase‐I activity in type 2 diabetes.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.