The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Platelet collagen receptors: a new target for inhibition?

Collagen is a major component of extracellular matrix and a wide variety of types exist. Cells recognise collagen in different ways depending on sequence and structure. They can recognise predominantly primary sequence, they may require triple-helical structure or they can require fibrillar structures. Since collagens are major constituents of the subendothelium that determine the thrombogenicity of the injured or pathological vessel wall, a major role is induction of platelet activation and aggregation as the start of repair processes. Platelets have at least two direct and one indirect (via von Willebrand factor) receptors for collagen, and collagen has specific recognition motifs for these receptors. These receptors and recognition motifs are under intensive investigation in the search for possible methods to control platelet activation in vivo. A wide range of proteins has been identified and, in part, characterised from both haematophageous insects and invertebrates but also from snake venoms that inhibit platelet activation by collagen or induce platelet activation via collagen receptors on platelets. These will provide model systems to test the effect of inhibition of specific collagen-platelet receptor interactions for both effectiveness as well as for side effects and should provide assay systems for the development of small molecule inhibitors. Since platelet inhibitors for long-term prophylaxis of cardiovascular diseases are still in clinical trials there are many unanswered questions about long-term effects both positive and negative. The major problem which still has to be definitively solved about these alternative approaches to inhibition of platelet activation is whether they will show advantages in terms of dose-response curves while offering decreased risks of bleeding problems. Preliminary studies would seem to suggest that this is indeed the case.

Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.

Effects of high-yield thrombocytapheresis on the quality of platelet products

BACKGROUND: The steadily increasing demands for single-donor apheresis platelet (PLT) concentrates (APCs) are a challenge to the PLT supply system. Therefore, efforts to improve plateletpheresis yield, allowing apheresis products to be split into 2 or more units, are valuable strategies. No data to demonstrate in vivo transfusion efficacy of these high-yield split-APCs are currently available, however. STUDY DESIGN AND METHODS: The transfusion efficacy of APCs produced by two apheresis methods involving different harvest and storing procedures and varying PLT yields was investigated. Efficacy measures were the 1-hour percent PLT recovery (PPR(1h)) and the 1-hour corrected count increment (CCI(1h)). In total, 400 APCs, produced with either an Amicus device (Baxter) and stored in PLT additive solution (T-Sol; Amicus method [AM], n = 107) or a Trima device (Gambro) and stored in plasma (Trima method [TM], n = 293), were transfused to 55 children (31 girls; median age, 9.5 years; range, 0.2-18.5 years) with thrombocytopenia due to chemotherapy or aplastic anemia (median, 4 APCs per child; range, 1-68). RESULTS: Transfusion efficacy was significantly lower for AM-APCs than for TM-APCs (median PPR(1h), 17 and 33%; median CCI(1h), 7.9 and 15.6, respectively; p < 0.001). Reduced transfusion efficacy correlated in a yield-dependent manner with high apheresis PLT yields (>/=6 x 10(11)) for AM-APCs (p < 0.001). CONCLUSION: Although in vitro validation of AM- and TM-APCs has been performed, only by evaluating transfusion efficacy in vivo did the AM turn out to be not suitable for high-yield thrombocytapheresis. This study recommends the implementation of in vivo transfusion efficacy studies for high-yield APC apheresis donations.

Diagnostic criteria for hematopoietic stem cell transplant-associated microangiopathy: results of a consensus process by an International Working Group

BACKGROUND AND OBJECTIVES: There are no widely accepted criteria for the definition of hematopoietic stem cell transplant -associated microangiopathy (TAM). An International Working Group was formed to develop a consensus formulation of criteria for diagnosing clinically significant TAM. DESIGN AND METHODS: The participants proposed a list of candidate criteria, selected those considered necessary, and ranked those considered optional to identify a core set of criteria. Three obligatory criteria and four optional criteria that ranked highest formed a core set. In an appropriateness panel process, the participants scored the diagnosis of 16 patient profiles as appropriate or not appropriate for TAM. Using the experts' ratings on the patient profiles as a gold standard, the sensitivity and specificity of 24 candidate definitions of the disorder developed from the core set of criteria were evaluated. A nominal group technique was used to facilitate consensus formation. The definition of TAM with the highest score formed the final PROPOSAL. RESULTS: The Working Group proposes that the diagnosis of TAM requires fulfilment of all of the following criteria: (i) >4% schistocytes in blood; (ii) de novo, prolonged or progressive thrombocytopenia (platelet count <50 x 109/L or 50% or greater reduction from previous counts); (iii) sudden and persistent increase in lactate dehydrogenase concentration; (iv) decrease in hemoglobin concentration or increased transfusion requirement; and (v) decrease in serum haptoglobin. The sensitivity and specificity of this definition exceed 80%. INTERPRETATION AND CONCLUSIONS: The Working Group recommends that the presented criteria of TAM be adopted in clinical use, especially in scientific trials.

Platelet GPIb complex as a target for anti-thrombotic drug development

Specific inhibition of platelet function is a major target of anti-thrombotic drug research. Platelet receptors are both accessible and specific but have multiple functions often linked to a wide range of ligands. GPIb complex is best known as a major platelet receptor for von Willebrand factor essential for platelet adhesion under high shear conditions found in arteries and in thrombosis. Recent animal studies have supported inhibition of GPIb as a good candidate for anti-thrombotic drug development with several classes of proteins showing important specific effects and the required discrimination between roles in haemostasis and thrombosis is important to protect against bleeding complications. These include antibodies, several classes of snake venom proteins, mutant thrombin molecules and peptides affecting subunit interactions. However, due to the nature of its receptor-ligand interactions involving large protein-protein interfaces, the possibility of developing classic pharmaceutical inhibitors for long term (and perhaps oral) treatment is still unclear, and additional information about structural interactions and signalling mechanisms is essential.

Preemptive analgesia by lornoxicam--an NSAID--significantly inhibits perioperative platelet aggregation

BACKGROUND AND OBJECTIVE: To investigate whether preemptive administered lornoxicam changes perioperative platelet function during thoracic surgery. METHODS: A total of 20 patients scheduled for elective thoracic surgery were randomly assigned to receive either lornoxicam (16 mg, i.v.; n = 10) or placebo (n = 10) preoperatively. All patients underwent treatment of solitary lung metastasis and denied any antiplatelet medication within the past 2 weeks. Blood samples were drawn via an arterial catheter directly into silicone-coated Vacutainer tubes containing 0.5 mL of 0.129 M buffered sodium citrate 3.8% before, 15 min, 4 h and 8 h after the study medication was administered. Platelet aggregation curves were obtained by whole blood electrical impedance aggregometry (Chrono Log). RESULTS: Platelet aggregation was significantly reduced 15 min, 4 h and 8 h after lornoxicam administration compared to placebo (P < 0.05) for collagen, adenosine diphosphate and arachidonic acid as trigger substances. Adenosine diphosphate-induced platelet aggregation decreased by 85% 15 min after lornoxicam administration, and remained impaired for 8 h. CONCLUSION: Platelet aggregation assays are impaired for at least 8 h after lornoxicam application. Therefore perioperative analgesia by use of lornoxicam should be carefully administered under consideration of subsequent platelet dysfunction.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.

Effects of cognitive behavioral stress management on HIV-1 RNA, CD4 cell counts and psychosocial parameters of HIV-infected persons

OBJECTIVE: To determine the effects of cognitive-behavioral stress management (CBSM) training on clinical and psychosocial markers in HIV-infected persons. METHODS: A randomized controlled trial in four HIV outpatient clinics of 104 HIV-infected persons taking combination antiretroviral therapy (cART), measuring HIV-1 surrogate markers, adherence to therapy and well-being 12 months after 12 group sessions of 2 h CBSM training. RESULTS: Intent-to-treat analyses showed no effects on HIV-1 surrogate markers in the CBSM group compared with the control group: HIV-1 RNA < 50 copies/ml in 81.1% [95% confidence interval (CI), 68.0-90.6] and 74.5% (95% CI, 60.4-85.7), respectively (P = 0.34), and mean CD4 cell change from baseline of 53.0 cells/microl (95% CI, 4.1-101.8) and 15.5 cells/microl (95% CI, -34.3 to 65.4), respectively (P = 0.29). Self-reported adherence to therapy did not differ between groups at baseline (P = 0.53) or at 12 month's post-intervention (P = 0.47). Significant benefits of CBSM over no intervention were observed in mean change of quality of life scores: physical health 2.9 (95% CI, 0.7-5.1) and -0.2 (95% CI, -2.1 to 1.8), respectively (P = 0.05); mental health 4.8 (95% CI, 1.8-7.3) and -0.5 (95% CI, -3.3 to 2.2) (P = 0.02); anxiety -2.1 (95% CI, -3.6 to -1.0) and 0.3 (95% CI, -0.7 to 1.4), respectively (P = 0.002); and depression -2.1 (95% CI, -3.2 to -0.9) and 0.02 (95% CI, -1.0 to 1.1), respectively (P = 0.001). Alleviation of depression and anxiety symptoms were most pronounced among participants with high psychological distress at baseline. CONCLUSION: CBSM training of HIV-infected persons taking on cART does not improve clinical outcome but has lasting effects on quality of life and psychological well-being.

Longitudinal platelet reactivity to acute psychological stress among older men and women

Platelet reactivity to acute stress is associated with increased cardiovascular disease risk; however, little research exists to provide systematic methodological foundations needed to generate strong longitudinal research designs. Study objectives were: 1) to evaluate whether markers of platelet function increase in response to an acute psychological stress test among older adults, 2) to establish whether reactivity remains robust upon repeated administration (i.e. three occasions approximately 1 year apart), and 3) to evaluate whether two different acute speech stress tasks elicit similar platelet responses. The 149 subjects (mean age 71 years) gave a brief impromptu speech on one of two randomly assigned topics involving interpersonal conflict. Blood samples drawn at baseline and post-speech were assayed using flow cytometry for platelet responses on three outcomes (% aggregates, % P-selectin expression, and % fibrinogen receptor expression). Three-level hierarchical linear modeling analyses revealed significant stress-induced increases in platelet activation on all outcomes (p < 0.001). No significant habituation on any measure was found. Additional reactivity differences were associated with male gender, history of myocardial infarction, and use of aspirin, statins, and antidepressants. The results demonstrate that laboratory acute stress tests continued to produce robust platelet reactivity on three activation markers among older adults over 3 years.

Effects of depressive and anxious symptoms on norepinephrine and platelet P-selectin responses to acute psychological stress among elderly caregivers

BACKGROUND: Caring for a spouse with Alzheimer's disease is associated with increased psychological distress, impaired immunity, and heightened cardiovascular risk. Hyperreactivity of sympathetic and platelet activation responses to acute psychological stress, or the failure to recover quickly from stressful events, may constitute an important pathway linking stress and negative affect with cardiovascular disease (CVD). OBJECTIVES: (1) To evaluate associations between negative affect (i.e., depressive and anxious symptoms) with increased norepinephrine and P-selectin responses to an acute psychological stress task. (2) To establish whether these associations are augmented among elderly spousal caregivers (CG) compared to non-caregivers (NC). METHODS: Depressive (DEP) and anxious (ANX) symptoms from the Brief Symptom Inventory were assessed among 39 CG and 31 NC. Plasma norepinephrine levels (NE) and percent platelet P-selectin (PSEL) expression were assayed at three time-points: rest, immediately following a laboratory speech test (reactivity), and after 14 min of recovery. Results: Among CG, but not NC, increased symptoms of depression and anxiety were associated with delayed NE recovery (DEP: beta=.460, p=.008; ANX: beta=.361, p=.034), increased PSEL reactivity (DEP: beta=.703, p<.001; ANX: beta=.526, p=.002), and delayed PSEL recovery (DEP: beta=.372, p=.039; ANX: beta=.295, p=.092), while controlling for age, gender, aspirin use, antidepressant use, and preexisting CVD. Bivariate correlations showed delayed NE recovery was also associated with increased PSEL reactivity (r=.416) and delayed PSEL recovery (r=.372; all ps<.05) among CG but not NC. DISCUSSION: Among chronically stressed caregivers, increased levels of depressive and anxious symptoms are associated with prolonged sympathetic activation and pronounced platelet activation. These changes may represent one pathway linking caregiving stress to cardiovascular risk.

Clinical outcomes after platelet transfusions in patients with thrombotic thrombocytopenic purpura

BACKGROUND: Reports of deterioration and death after platelet (PLT) transfusions in patients with thrombotic thrombocytopenic purpura (TTP) have led to recommendations that they should not be given except for life-threatening hemorrhage. STUDY DESIGN AND METHODS: Published reports of PLT transfusions in patients with TTP were systematically reviewed and data from the Oklahoma TTP-HUS Registry, an inception cohort of 382 consecutive patients, 1989 through 2007, were analyzed. RESULTS: A systematic review identified 34 publications describing outcomes of patients with TTP after PLT transfusions: 9 articles attributed complications to PLT transfusions, 4 suggested that they may be safe, and 21 articles did not comment about a relation between PLT transfusions and outcomes. Fifty-four consecutive patients from the Oklahoma TTP-HUS Registry were prospectively analyzed. ADAMTS13 activity was less than 10 percent in 47 patients; also included were 7 patients whose activity was not measured but who may have been deficient. Thirty-three (61%) patients received PLT transfusions. The frequency of death was not different between the two groups (p = 0.971): 8 (24%) patients who received PLT transfusions died (thrombosis, 5; hemorrhage, 1; sepsis, 2) and 5 (24%) patients who did not receive PLT transfusions died (thrombosis, 4; hemorrhage, 1). The frequency of severe neurologic events was also not different (p = 0.190): 17 (52%) patients who received PLT transfusions (in 5 of these 17 patients, neurologic events only occurred before PLT transfusions) and 7 (33%) patients who did not receive PLT transfusions. CONCLUSION: Evidence for harm from PLT transfusions in patients with TTP is uncertain.