ALBERT

Description: Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selective T-cell subsets. It is also a major component of the endothelial cell intercellular junction. Previous studies have shown that cross-linking PECAM-1 on the surface of leukocytes results in the activation of adhesion molecules of both the β1 and β2integrin family. In addition, the process of leukocyte transendothelial migration appears to be mediated, at least in part, by homophilic adhesive interactions that take place between leukocyte and endothelial cell junctional PECAM-1 molecules. However, little is known about the functional role of this membrane glycoprotein in human platelets. In the present study, we examined the effects of PECAM-1 engagement on integrin-mediated platelet-extracellular matrix or platelet-platelet interactions. Bivalent, but not monovalent, anti–PECAM-1 monoclonal antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface coverage) and aggregation (increased average size) onto extracellular matrix, under both oscillatory or defined low shear flow conditions (200 s−1) in a modified cone and plate viscometer. Moreover, bivalent anti-domain 6 MoAbs were capable of serving as costimulatory agonists to markedly enhance both adenosine diphosphate (ADP)- and platelet activating factor (PAF)-induced platelet aggregation responses. These antibodies appeared to act via outside-in signal transduction through PECAM-1, as evidenced by the fact that their binding (1) led to conformational changes in the αIIbβ3 integrin complex, (2) induced surface expression of P-selectin, and (3) resulted in the tyrosine phosphorylation of PECAM-1. Together, these data support a role for PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a costimulatory agonist receptor capable of modulating integrin function in human platelets during adhesion and aggregation.

Description: Diagnosis of platelet function disorders is based on the nature of bleeding symptoms, a positive family history and abnormal laboratory tests. However, currently available methods are time and labour consuming and lack sensitivity and specificity. In the present study we evaluated the performance of the Cone and Plate (let) analyzer (CPA) for screening patients with a suspected platelet function disorder and for monitoring therapy. Consecutive patients presenting with spontaneous or post surgical bleeding were studied. All patients underwent primary and secondary hemostasis evaluation including CPA testing. In this test a volume of 200μL of citrated whole blood is placed onto polystyrene wells and submitted to flow (shear rate of 1800 sec. −1) by a Teflon cone for 2 minutes. The resulting platelet deposition is then read by an image analyzer and expressed as % of the well surface covered ( SC, %) with adhered platelets and as the average size (AS, μm2)of platelet aggregates. Patients with a platelet function disorder were further tested with a modified CPA test in which blood samples are pre-incubated (1 min.) with a sub-optimal concentration of an agonist prior to subjecting the sample to the regular CPA test. Response of platelets to the agonist leads to agglutination and micro aggregates formation in the suspension phase of the test tube with consequent decline in platelet adhesion. 165 consecutive bleeding patients were evaluated and compared to 54 healthy controls. We diagnosed 51 patients as suffering from a platelet function disorder, of whom 8 patients (GTA – 7, BSS – 1) were excluded. The remaining 43 patients presented complete unresponsiveness to epinephrine in repeated platelet aggregation tests while all other coagulation, liver and renal function tests were normal and similar to healthy controls. In the CPA test these 43 patients had a significantly lower SC values compared to the other patients or to healthy controls. To further explore the CPA’s diagnostic capability 6 patients were compared to 16 randomly chosen controls using the modified CPA test. Whereas SC declined significantly by 73% in healthy controls after pre-incubation with epinephrine a non significant decline of only 15% was noted in patients with platelet adrenergic receptor dysfunction (table). Seventeen patients with platelet adrenergic receptor dysfunction underwent surgery. Following pre-surgical administration of DDAVP SC values followed closely and the VWF:RCo levels whereas the response to epinephrine in routine aggregometry did not change. All patients underwent successful operations without excessive bleeding. Our data suggest that patients with adrenergic receptor dysfunction suffer from a combined aggregation and adhesion platelet defects. The CPA test was found useful for screening both adhesion and aggregation of platelet disorders including adrenergic receptor dysfunction as well as for monitoring DDAVP therapy in these patients. Adrenergic receptor dysfunction No disorder Healthy controls * max. response to epinephrine in aggregometry ** 1 min. incubation with epinephrine N 43 114 54 Bleeding time (min.) 5.6±2.5 5.5±2.3 6.7±3.1 Aggregomatry * 23% 82% 81% SC% 6.2±2.4 11.5±3.4 12.3±3.0 Modifioed CPA-pre 6.5±2.2 9.4±1.8 Modified CPA-post** 5.4±1.3 2.5±0.8

Description: The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) that is released from endothelial cells as a large multimeric protein. However, the specific physiological conditions required for the function of this von Willebrand factor-cleaving protease (VWF-CP) are not yet established. In this study we determined the optimal conditions for the VWF-CP activity using the Cone and Plate(let) Analyzer (CPA). Proteolysis of a soluble recombinant VWF by a recombinant VWF-CP in the absence or the presence of BaCl2 (for induction of VWF-CP) was performed under static and flow (2050 s−1) conditions. The resulted fragments of the soluble VWF were immobilized on a polystyrene surface and non-adhering sites were blocked with 1% BSA. In parallel, polystyrene surface immobilized VWF was similarly treated by VWF-CP. The immobilized cleaved VWF fragments by the above protocols served as a substrate for citrated whole blood platelet adhesion under flow (2050 s−1). Reduction of platelet adhesion (surface coverage, SC) in BaCl2 treated compared to untrerated sample reflects the degree of VWF-CP activity. Platelet adhesion (SC, %) to VWF proteolytic products generated under the indicated conditions: Static w/o BaCl2 Static + BaCl2 Flow w/o BaCl2 Flow + BaCl2 Soluble VWF 15.0 ± 3.4 11.1 ± 2.7 13.6 ± 2.2 8.0 ± 2.1 Immobil. VWF 19.1 ± 3.0 12.0 ± 4.1 6.5 ± 1.1 1.0 ± 0.6 Maximal VWF-CP activity as reflected by maximal reduction (85%) of platelet adhesion was observed when immobilized VWF was treated by VWF-CP under flow. Minimal effect of VWF-CP activity was observed when soluble VWF was treated under static condition (26% reduction). Intermediate effect was observed with soluble VWF under flow (41%) and with immobilized VWF under static condition (37%). It should be noted that in the case of immobilized VWF application of flow alone (without BaCl2) was sufficient to induce a remarkable decrease of platelet adhesion (66%). In conclusion, both immobilization of VWF and high shear stress are important conditions for VWF-CP function, suggesting that stretching of immobilized VWF under flow exposes the VWF-cleavage site and thereby may serve as a control mechanism of platelet adhesion at the site of thrombus formation.

Description: Background: In patients with stable coronary artery disease, and patients undergoing elective percutaneous coronary intervention (PCI), laboratory resistance to aspirin is associated with a higher incidence of adverse events. Nevertheless, the responsiveness to aspirin in acute myocardial infarction (AMI) and its implications have not yet been investigated. Methods: The study comprised 76 aspirin naïve patients who underwent primary PCI (PPCI) for ST-elevation MI (STEMI). Platelet reactivity was assessed 30–60 mins after a loading dose of 300mg chewable aspirin, by conventional aggregometry and Impact R, where platelet reactivity to arachidonic acid (AA) was expressed by platelet deposition under flow conditions. Results: Patients were stratified using the median value of AA-induced platelet aggregation (PA) (49%) to good responders to aspirin (n=38), who had a median AA-induced PA of 33% (25–41), and poor responders to aspirin (n=38), who had a median AA-induced PA of 77% (70–84). Similarly, good compared with poor responders had higher surface coverage by Impact R (3.9±2.6 vs. 2.2±1.3, p=0.003). Good versus poor responders were similar regarding baseline demographic, clinical and angiographic characteristics. However, good responders were more likely to demonstrate early ST-segment resolution ≥ 70% after PPCI (84% vs 54%, p

Description: Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selective T-cell subsets. It is also a major component of the endothelial cell intercellular junction. Previous studies have shown that cross-linking PECAM-1 on the surface of leukocytes results in the activation of adhesion molecules of both the β1 and β2integrin family. In addition, the process of leukocyte transendothelial migration appears to be mediated, at least in part, by homophilic adhesive interactions that take place between leukocyte and endothelial cell junctional PECAM-1 molecules. However, little is known about the functional role of this membrane glycoprotein in human platelets. In the present study, we examined the effects of PECAM-1 engagement on integrin-mediated platelet-extracellular matrix or platelet-platelet interactions. Bivalent, but not monovalent, anti–PECAM-1 monoclonal antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface coverage) and aggregation (increased average size) onto extracellular matrix, under both oscillatory or defined low shear flow conditions (200 s−1) in a modified cone and plate viscometer. Moreover, bivalent anti-domain 6 MoAbs were capable of serving as costimulatory agonists to markedly enhance both adenosine diphosphate (ADP)- and platelet activating factor (PAF)-induced platelet aggregation responses. These antibodies appeared to act via outside-in signal transduction through PECAM-1, as evidenced by the fact that their binding (1) led to conformational changes in the αIIbβ3 integrin complex, (2) induced surface expression of P-selectin, and (3) resulted in the tyrosine phosphorylation of PECAM-1. Together, these data support a role for PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a costimulatory agonist receptor capable of modulating integrin function in human platelets during adhesion and aggregation.

Description: Red blood cells (RBC) contribute significantly to hemostasis and thrombosis under oscillatory flow conditions, and erythrocytosis has been associated with increased thrombotic risk. To capture dynamic effects of RBC on platelets, we used a recently described Cone and Plate (let) Analyzer (CPA), to evaluate the effect of hematocrit (Hct) on platelet function in whole blood under arterial flow conditions (1800 sec-1, 2 min, 25°C). Anticoagulated blood, reconstituted to varying hematocrits with autologous RBC, demonstrated a significant increase (30–50%, n= 12, p 〈 0.05) in adherent platelet aggregate size at Hct levels 〉45%. No significant effect on platelet adhesion was noted. Increases in aggregate size were not accompanied by significant platelet dense or alpha granule secretion, measured using 14C-serotonin release and Platelet Factor 4 ELISA assays, respectively. However, when cell-free supernatants (SNT) were prepared post shearing to investigate the presence of proaggregatory activity by adding SNT to fresh PRP (1:10 SNT: PRP ratio), and quantifying the formation of small platelet aggregates with a laser light scatter aggregometer, SNT derived from sheared high Hct samples contained nearly twice (1.75 + 0.16, n=5, p 〈 0.05) the activity of sheared normal Hct samples. The erythrocytosis-induced increase in platelet aggregate size was not affected by pretreatment of blood with 0.05 mM aspirin, but could be prevented completely by antagonism of P2Y1 (MRS2179, 200 μM), P2Y12 (2-methylthioadenosine 5′-monophosphate, 10–100 μM), or P2X1 (MRS2159, 200 μM), ADP and ATP receptors, respectively, as well as by converting exogenous ADP to ATP with a combination of creatine phosphate (2 mM) and creatine phosphokinase (20 U/ml). Whereas negligible platelet granule secretion was measured post shear, metabolic inhibition of RBC with 1% sodium azide or 0.25% glutaraldehyde fixation (2 h, 37°C) fully inhibited erythrocytosis-enhanced increases in platelet aggregate size. Thus, the adenine nucleotides contributing to erythrocytosis-enhanced platelet aggregate formation under physiologic shear appear to be derived predominantly from RBC. Taken together, the data suggest that ADP and ATP are both required for erythrocytosis-induced large platelet aggregate formation, but that the presence of ATP alone is not sufficient. The results may further suggest a role for direct ADP or ATP receptor blockade for the prevention of erythrocytosis-associated thrombotic complications.

Description: Background Von Willebrand disease (VWD) is the most common significant bleeding disorder in the western world. VWD presents with a wide spectrum of clinical manifestations, ranging from totally asymptomatic individuals to severe, occasionally life-threatening bleeding episodes and hence early and accurate diagnosis is crucial for starting treatment. Diagnosis of VWD requires specialized laboratories with skilled personnel and the testing process itself is time and labor consuming, and is costly. In recent years several methods for VWD diagnosis were developed including several flow bedside instruments which test whole blood coagulation following shear stress activation of clotting. One such tool is the cone and plate(let) analyzer (CPA). In the CPA test whole blood is activated by a rotating conical disc on a polystyrene surface at high shear rate. The test result is expressed as the % of the plastic surface covered with adherent platelets (SC- surface coverage %) and the average size of the platelet aggregates (average size- AS μm2). The aim of the current study was to test the properties of the CPA as a diagnostic tool for VWD in a non-selected group of patients, to compare its diagnostic abilities with current methods of diagnosis, and to test for presence of (patient and physician) bias in the diagnostic process. Methods Patients referred for evaluation of bleeding symptoms, post-surgical bleeding, abnormal coagulation tests or with a known familial bleeding disorder were compared to a group of healthy controls and warfarin treated patients. Diagnosis of VWD and all other bleeding disorders was achieved by standard clinical and laboratory methods and in the final analysis VWD patients were compared to all other non VWD groups. The CPA was tested in all patients and controls, first on an inception cohort (268 subjects) and results from that sample were then applied to a validation cohort (221 subjects). The optimal SC and AS values were determined by ROC analysis in each sample and both samples overall. Sensitivity, specificity and likelihood ratios (likelihood of having (LR+) or not having (LR-) VWD given a specific cut-off value) were computed for each cut-off value. ROC method was also applied to test whether age, gender, severity of VWD, presence of bleeding symptoms, genetic/familial diagnosis of VWD and comorbidity of patients had any effect on CPA's testing accuracy. Results During a 10 year period 489 consecutive patients referred to Sheba medical Center were studied. Following a complete workup we diagnosed (in both cohorts) 149 patients with VWD (type 1-72, type 2-42, type 3+acquired VWD- 35), that were compared to 217 healthy controls, 35 VKA treated patients, and 88 patients with other bleeding disorders (hemophilia A/B – 39, Factor II, VII, XI deficiencies – 49). The surface coverage was found as the best CPA diagnostic parameter for diagnosis of VWD, with an optimal cut-off value of 8.3% in the inception cohort and 6.9 % (but with better sensitivity) in the validation cohort (table and figure). In all of the study cohorts SC optimal cut-off maintained its high specificity with an improved sensitivity and a cut-off value of 7.1%. Age, sex, severity of VWD, or additional genetic/familial diagnosis of VWD had no effect on CPA's testing accuracy. However, patients tested soon after a bleeding event or during hospitalization (originated mainly from the validation cohort) were older, had higher levels VW activity and higher levels of SC and therefore different SC cut-off value with different test characteristics (table). Conclusion In this relatively large and representative cohort of healthy volunteers and patients with various bleeding disorders the CPA test was found sensitive and highly specific in the diagnosis of VWD. The test was robust to patient's age, sex, and VWD severity but a mild spectrum bias cannot be ruled out (as found in the validation cohort) deriving mainly from patient's comorbidity and recent bleeding. This highly specific test may prove its utility in preliminary or urgent diagnosis of VWD before a complete clinical and laboratory workup is done. Disclosures: Savion: Mattis medical: Equity Ownership.

Description: Recent studies established that platelets, beside their haemostatic function play a role in inflammation, immunity and atherosclerosis. In this study, we investigated the role of platelets in CD4+ lymphocyte adhesion to the subendothelial extracellular matrix (ECM) under static and flow conditions. Cultured clone of CD4+ lymphocyte (HVS infected clone) was incubated for 30 min at 37°C under static or flow conditions on tissue culture plates pre-coated with ECM, in the presence or absence of gel-filtered platelets. In separate experiments, the ECM plates were further coated with platelet-poor (PPP) or platelet-rich plasma (PRP) prior to the addition of cells. Flow conditions were applied using the Cone and Plate(let) Analyzer (CPA) device and the results were evaluated by an image analysis system and expressed as the number of adhered T-cells per mm2. Under static conditions, activation of T-cells with phorbol 12-myristate 13-acetate (PMA) led to 2.6-fold increase in cell adhesion. In the presence of platelets the adhesion rate did not change. In contrast, under flow condition (200 s−1), platelets substantially enhanced both resting and PMA-activated T-cells adhesion (12.7- and 18.5-fold, respectively). Moreover, under flow, platelet-T-cell heterotypic clusters appeared on the ECM as revealed by both light and scanning electron microscopy. When ECM was pre-coated with PPP, adhesion of T-cells was enhanced by 2.3- and 1.8-fold under static and flow conditions, respectively. Pre-coating with PRP did not change T-cells adhesion under static but further enhanced T-cells adhesion under flow (by 80% vs. PPP). In this case platelet-lymphocyte clusters were not observed. Similar pattern of results was seen with natural blood-derived CD4+ lymphocytes cultured for 7 days. In these natural T-cells the adhesion under both static and flow conditions was platelet dependent, however, higher adhesion under flow compared to static conditions (55-fold) due to relatively low T-cells adhesion under static conditions and to the heterotypic cluster formation occurring only under flow conditions was observed. We further investigated the role of different receptors in T-cell-platelet cluster formation on ECM. Blockade of β1-dependent integrins on T-cells was followed by a decrease in adhesion under both static and flow conditions in the presence of platelets (by 37% and 43%, respectively). Combined blockade of CD40 ligand (CD40L) and P-selectin glycoprotein ligand-1 (PSGL-1) receptors on the T-cells decreased their adhesion in the presence of platelets by 72% under high shear (600 s−1) but not under low shear (200 s−1) and under static conditions. Blockage of the platelet integrin αIIbβ3 by tirofiban markedly reduced cluster formation thereby decreasing T-cells adhesion (by 90%) under flow conditions. The results of this study show that platelets support CD4+ lymphocyte adhesion to ECM under flow conditions by formation of heterotypic clusters that are dependent on platelet adhesion and aggregation and mediated by CD40L, PSGL-1 and β1-dependent integrins.

Description: Measurement of von Willebrand factor-cleaving protease (VWF-CP) activity is useful for diagnosis of thrombotic thrombocytopenic purpura (TTP), but the current methods are cumbersome. We recently introduced a simple method of TTP detection using the Cone and Plate(let) Analyzer (CPA), that was based on the observation that mixing of acute TTP plasma (50 μL) with normal whole blood (150 μL) induces a significant increase in platelet deposition (surface coverage, %SC) on polystyrene surface under flow condition (1800 s−1) (Brit J Haematol, 120: 597, 2003). The test was specific for TTP, since plasma from patients with HUS, APLS, ITP or HIT did not affect normal platelet deposition. In this study, we further explored the potential use of the CPA method in differential diagnosis of inherited and acquired TTP by increasing the shear rate and by inducing the VWF-CP activity with BaCl2. TTP or normal plasma was mixed with normal blood (type O+) and the surface covered by platelets was tested under shear rates ranging between 1800 s−1 to 2500 s−1. Maximal difference in SC between TTP and normal plasma was observed at a shear rate of 2050 s−1. Under these conditions, plasmas of 5 patients with inherited TTP (ITTP), and 11 patients with acquired TTP (ATTP) yielded comparable increase in SC of normal platelets in whole blood, i.e. 77±19% and 78±17%, respectively. However, experiments in which BaCl2 was added to allow activation of VWF-CP resulted in a substantial distinction between inherited and acquired TTP plasma. While addition of BaCl2 to normal or inherited TTP plasma, both mixed with normal blood, yielded similar reductions of SC by 41±12% and 51±19%, only 11±4% reduction of SC was observed when acquired TTP plasma was used. These results suggest that when normal or inherited TTP plasma is mixed with normal blood, activation of VWF-CP cleaves large VWF multimers giving rise to substantial reduction in SC, whereas in the case of acquired TTP plasma the inhibitor of VWF-CP abolishes BaCl2-induced activation of VWF-CP and the ensuing reduction in SC. We conclude that introduction of BaCl2 in the CPA at high shear conditions may be useful for differentiation between inherited and acquired TTP.

Description: Platelet anti-aggregants play a major role in the treatment of cardiovascular disorders. Recently it has been suggested that a significant proportion of patients fail to respond to these agents according to different testing methods. We have developed a new method for evaluating the response to platelet anti-aggregants, the modified Cone and Plate(let) Analyzer (CPA) test. The method is based on the observation that pre-activation of platelets by an agonist will result in reduced platelet deposition on a polystyrene surface under arterial flow conditions (the phenomenon of platelet adhesion refractoriness). In vitro studies demonstrated that the basic platelet adhesion (surface coverage, SC 12.3±6.8%) was significantly reduced in response to pre-incubation of the sample (for 1 min) with arachidonic acid (AA, SC 2.1±1.5%), adenine diphosphate (ADP, SC 1.3±0.6%) and epinephrine (EPI, 2.9±0.9%). This effect was selectively inhibited by aspirin (for the response to AA, SC 8.1±3.8%), and by 2-Methylthio-AMP triethylammonium (2-MeSAMP), a selective inhibitor of P2Y12 (for the response to ADP, SC 4.8±2.0%), p