scholarly journals Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

2021 ◽  
Author(s):  
Alexey Martyanov ◽  
Ivan Tesakov ◽  
Olga An ◽  
Julia-Jessica Korobkin ◽  
Anastasia Ignatova ◽  
...  
Keyword(s):  
Parallel Plate ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Lower Boundary ◽  
Calcium Mobilization ◽  
Control Group ◽  
Functional Responses ◽  
Kaposiform Hemangioendothelioma ◽  
Normal Ranges ◽  
Platelet Receptor

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from six patients with KHE and one KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, their CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

scholarly journals Characterization of platelet functionality in pediatric patients with kaposiform hemangioendothelioma / Kasabach-Merritt phenomenon

2021 ◽  
Author(s):  
Alexey Martyanov ◽  
Ivan Tesakov ◽  
Olga An ◽  
Julia-Jessica Korobkin ◽  
Anastasia Ignatova ◽  
...  
Keyword(s):  
Parallel Plate ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Lower Boundary ◽  
Calcium Mobilization ◽  
Control Group ◽  
Functional Responses ◽  
Kaposiform Hemangioendothelioma ◽  
Normal Ranges ◽  
Platelet Receptor

Background. Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy commonly associated with Kasabach-Merritt phenomenon (KMP) that includes thrombocytopenia and coagulation dysfunction. Platelet receptor CLEC-2 -tumor cell podoplanin interaction is considered the key mechanism of thrombocytopenia in KMP, however, the effect of long-term exposure to podoplanin on platelet function is unknown. Procedure. Here we examined blood samples from 7 patients with KHE/KMP. Platelet calcium signaling and functional responses to conventional activation and CLEC-2 stimulation were analyzed by continuous and endpoint live cell flow cytometry. Platelet aggregation in response to ADP or rhodocytin was analyzed by low-angle light scattering approach (LaSca). Additionally, ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Results. We demonstrate that in KHE/KMP platelet functional responses to strong stimulation were on the lower boundary of age-matched normal ranges, while calcium mobilization and fibrinogen binding upon stimulation with ADP alone were significantly lower than control values. Platelet di-aggregate formation in response to ADP was also diminished in most of the patients. Formation of platelet aggregates in collagen-coated parallel plate flow chambers was also noticeably lower than in the age-matched control group. Calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients and could be blocked by low-molecular-weight inhibitors, 2CP and HB125. Conclusions. While platelet responsiveness in KHE/KMP is moderately altered, platelet CLEC-2 receptors remain functional and respond to inhibition. Therefore, our findings suggest that CLEC-2-targeting molecules are new potential agents in therapeutic management of this life-threatening condition.

Inhibition of Platelet Glycoprotein β3 by Chimeric Monoclonal Antibody Prevents Arterial Thrombosis in Beagle Dogs,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3357-3357
Author(s):  
Shundong Ji ◽  
Miao Jiang ◽  
Ningzheng Dong ◽  
Xia Bai ◽  
Changgeng Ruan
Keyword(s):  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Dose Range ◽  
Negative Control Group ◽  
Negative Control ◽  
Platelet Glycoprotein ◽  
Control Group ◽  
Beagle Dogs

Abstract Abstract 3357 In this present study in beagle dogs, we evaluated the antithrombotic efficacy of Pulaimab, the chimeric monoclonal antibodies SZ21-F(ab)2 fragments against platelet glycoprotein (GP) β3, in a modified Folts model. The Folts model is widely accepted to be effective and clinically relevant for testing potential anti-thrombotic agents, in this model the cyclic flow reductions (CFRs) are caused by platelet dependent thrombi that form under high-shear conditions at injured stenosed sites of an artery. Thirty beagle dogs of either sex, weighing 7.5 to 14 kg, were randomly divided into five groups of six (three females and three males), the details were following: negative control group (injected normal saline), positive control group (injected 0.2mg/Kg of Reopro), and three experimental groups [injected 0.2, 0.4, 0.8 mg/Kg of SZ21-F(ab)2, respectively]. A dose range from 0.2 to 0.8 mg/kg of Pulaimab significantly reduced the CFRs by 21–73%, without reduction of platelet numbers and prolongation of the bleeding time. Ex vivo ADP-induced platelet aggregation was equally reduced. The present study demonstrates that the inhibition of platelet GP αIIbβ3function by SZ21-F(ab)2 is a powerful intervention to prevent platelet thrombus formation in injured arteries without thrombocytopenia and prolongation of the bleeding time. We therefore conclude that F(ab)2 fragments of inhibitory anti-GPβ3 antibodies may be useful compounds to prevent thrombosis. Disclosures: No relevant conflicts of interest to declare.

The Effect of Platelet PlA Polymorphism on Experimental Thrombus Formation in Man Depends on Blood Flow and Thrombogenic Substrate

2001 ◽  
Vol 85 (06) ◽  
pp. 1097-1103 ◽  
Author(s):  
Kjell Sakariassen ◽  
Hélène Grandjean ◽  
Claire Thalamas ◽  
Bernard Boneu ◽  
Pierre Sié ◽  
...  
Keyword(s):  
Blood Flow ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Flow Properties ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Platelet Receptor ◽  
Platelet Deposition ◽  
Coronary Syndromes ◽  
Platelet Thrombus

SummaryA number of studies have reported conflicting data on the association of the PlA1/PlA2 polymorphism of the GPIIIa gene and coronary syndromes. We have investigated the effect of this polymorphism on experimental platelet thrombus formation in man. Forty healthy male volunteers were genotyped for the PlA1/PlA2 polymorphism. Thrombus formation was induced ex vivo by exposing a tissue factor (TF) or a collagencoated coverslip in a parallel plate perfusion chamber to native blood for 2 and 4 min. The shear rates at these surfaces were 650 and 2,600 s–1. Platelet and fibrin deposition was quantified by immunoenzymatic methods. The frequencies of PlA1/PlA1 and PlA1/PlA2 genotypes were 52.5% and 47.5%, respectively. Ex vivo deposition of fibrin on TF was not affected by the PlA1/PlA2 polymorphism. However, the ex vivo platelet deposition at 650 s–1 was higher in blood from PlA1/PlA1 individuals than in PlA1/PlA2 individuals (P = 0.008 at 4 min). On collagen, neither fibrin nor platelet deposition was significantly affected by the PlA1/PlA2 polymorphism. Platelet thrombus formation is significantly influenced by genetic variations in the GPIIIa platelet receptor. This effect depends on the blood flow properties and the nature of the thrombogenic stimulus.

Blood Interaction with a Bioline Heparin Coated HIA-VAD: A Study on Calves

1997 ◽  
Vol 20 (1) ◽  
pp. 43-50 ◽  
Author(s):  
K.W.H.J. Van Der Kamp ◽  
C.P.E. Magielse ◽  
J.M. Elstrodt ◽  
J. Van Der Meer ◽  
W. Van Oeveren ◽  
...  
Keyword(s):  
Blood Cell ◽  
Degradation Products ◽  
Blood Compatibility ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Ventricular Assist Devices ◽  
Circulatory Support ◽  
Mechanical Trauma ◽  
Control Group ◽  
Implantation Procedure

The blood compatibility of ventricular assist devices developed by the Helmholtz Institute Aachen (HA-VAD's) was tested on calves. Seven calves received a non-coated HIA-VAD (control) and three a Bioline heparin coated device. The circulatory support of these HIA-VAD's lasted one week. Mechanical blood cell trauma estimated by hematocrit (Hct), hemoglobin (total Hb) and free plasma hemoglobin (free Hb) levels did not differ in either group. All HIA-VAD's in the control group remained thrombus free, except on one occasion when an inflow cannula was obstructed by a thrombus located in the tip. After circulatory support, the animals in this group seemed clinically healthy. However, thrombus formation was observed in the three heparin coated HIA-VAD's. One animal in this group died from complications after re-operation for pneumothorax on the fifth day of support, whereas the other two animals seemed clinically healthy. In these three animals, a stronge decrease in platelet numbers was measured even after 24 hours of support which recovered after 72 hours. This decrease in platelet numbers was associated with a lower degree of platelet aggregation ability stimulated by ADP (p<0.05). Fibrin(ogen) degradation products (FDP) increased significantly immediately after the implantation procedure (p<0.05). Fibrinogen levels initially decreased during the implantation procedure, but increased thereafter in both groups. The FDP levels remained high in this group, although the FDP levels in both groups were decreased after the implantation procedure. The ex vivo measured circulating heparin levels were lower in the heparin coated HIV-VAD group despite the equally administrated heparin doses in both animal groups. No differences were measured in either group with regard to white blood cell (WBC) numbers and complement hemolytic activity (CH50). Despite these hemostatic changes, no mechanical trauma could be demonstrated after seven days of circulatory support.

The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons

2008 ◽  
Vol 100 (10) ◽  
pp. 670-677 ◽  
Author(s):  
Alexandre Fontayne ◽  
Muriel Meiring ◽  
Seb Lamprecht ◽  
Jan Roodt ◽  
Eddy Demarsin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Side Effects ◽  
Blood Loss ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Platelet Glycoprotein ◽  
Fab Fragment ◽  
Platelet Receptor

SummaryThe Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibα and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

IN VIVO CHANGES IN SYSTEMIC PCO2 AND PO2 INFLUENCE THE THROMBOEMBOLIC REACTION FOLLOWING WALL PUNCTURE IN VENULES BUT NOT IN ARTERIOLES

1987 ◽  
Author(s):  
Mirjam G A oude Egbrink ◽  
Geert Jan Tangelder ◽  
Dick W Slaaf ◽  
Robert S Reneman
Keyword(s):  
Thrombus Formation ◽  
Fluid Dynamic ◽  
Blood Platelets ◽  
Control Group ◽  
Blood Gas ◽  
Systemic Blood ◽  
Normal Ranges ◽  
Experimental Group

Changes in pH and PCO2 influence the aggregation of blood platelets in response to various agents in vitro. In the present study intravital video-microscopy was used to investigate whether changes in systemic blood gas values influence the thromboembolic reaction in vivo as induced by vessel wall injury.The microtrauma was induced by puncturing the walls of microvessels in the rabbit mesentery (diameter range: 20-40 μm) with glass micropipets (tip diameters: 6-8 μm). The thromboembolic reactions were compared in two groups of anesthetized rabbits. The control group was ventilated to keep the blood gas values within normal ranges (means: pH=7.40, pCO2=32.9 mmHg, pO2=104.7 mmHg). The experimental group breathed spontaneously (mean blood gas values: pH=7.34, pCO2=50.5 mmHg, pO2=48.1 mmHg). The pCO2 and pO2 values were significantly different between both groups.In arterioles and venules of both groups bleeding and thrombus formation started immediately following wall puncture. Bleeding times were short (medians between 1.0 and 2.6 s). Parts of the thrombi started to embolize between 11.4 and 18.2 s following wall puncture (medians). In the control group embolization continued for 101 s in the arterioles and 17 s in the venules; during these periods 6 and 1 emboli were produced, respectively (all median values). In the experimental group the duration of embolization in the arterioles was 143 s in which period 7.5 emboli were produced, values not significantly different from control. In the venules of the experimental group embolization and hence platelet reaction went on uninhibited during the whole observation period of 600 s and 30 emboli were produced. Fluid dynamic factors cannot explain the differences in thromboembolic reaction between the control and experimental venules; vessel diameters and red blood cell velocities were not significantly different between both groups. Therefore, it is likely that the change in thromboembolic reaction in the venules results from the changes in systemic PCO2 and/or pO2. The different reactions in arterioles and venules in response to the altered systemic blood gas values might arise from different reactions in the vessel walls.

scholarly journals Exposure to Perfluoroalkyl Chemicals and Cardiovascular Disease: Experimental and Epidemiological Evidence

2021 ◽  
Vol 12 ◽  
Author(s):  
Alessandra Meneguzzi ◽  
Cristiano Fava ◽  
Marco Castelli ◽  
Pietro Minuz
Keyword(s):  
Cardiovascular Disease ◽  
Cardiovascular Events ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Consumer Products ◽  
Vascular Lesions ◽  
Calcium Signal ◽  
Glucose Metabolism Disorders ◽  
Platelet Response ◽  
Functional Responses

Polyfluoro- and perfluoro–alkyl substances (PFAS) are organic chemicals extensively used worldwide for industry and consumer products. Due to their chemical stability, PFAS represent a major cause of environmental pollution. PFAS accumulate in animal and human blood and tissues exerting their toxicity. We performed a review of the epidemiological studies exploring the relationship between exposure to PFAS and thromboembolic cardiovascular disease. An increase in cardiovascular disease or death related to PFAS exposure has been reported from cross-sectional and longitudinal observational studies with evidence concerning the relation with early vascular lesions and atherosclerosis. Several studies indicate an alteration in lipid and glucose metabolism disorders and increased blood pressure as a possible link with cardiovascular thromboembolic events. We also examined the recent evidence indicating that legacy and new PFAS can be incorporated in platelet cell membranes giving a solid rationale to the observed increase risk of cardiovascular events in the populations exposed to PFAS by directly promoting thrombus formation. Exposure to PFAS has been related to altered plasma membrane fluidity and associated with altered calcium signal and increased platelet response to agonists, both in vitro and ex vivo in subjects exposed to PFAS. All the functional responses are increased in platelets by incorporation of PFAS: adhesion, aggregation, microvesicles release and experimental thrombus formation. These findings offer mechanistic support the hypothesis that platelet-centred mechanisms may be implicated in the increase in cardiovascular events observed in populations chronically exposed to PFAS.

scholarly journals Observation of granulocyte function during ex vivo thrombus formation for patients with ANKRD26-associated thrombocytopenia

2020 ◽  
Vol 19 (1) ◽  
pp. 27-34
Author(s):  
D. S. Morozova ◽  
A. A. Martyanov ◽  
M. A. Panteleev ◽  
P. A. Zharkov ◽  
D. V. Fedorova ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Published Data ◽  
Platelet Activity ◽  
Functional Responses ◽  
Movement Velocity ◽  
Plane Flow ◽  
Healthy Donors

ANKRD26-associated thrombocytopenia is a non-syndromic hereditary thrombocytopenia for which there are currently no formal diagnostic criteria. It is known that the probability of myeloid leukemia in patients with pathogenetic variants in the ANKRD26 gene significantly increases, however, studies of the functioning of granulocytes in this pathology have not been conducted. Aims: Analysis of the functioning of granulocytes and platelets during ex vivo thrombosis in patients with ANKRD26-associated thrombocytopenia. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Two patients and 10 healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed by continuous flow cytometry. Ex vivo thrombus formation and granulocyte functioning were observed on a fluorescence microscope in parallel-plane flow chambers containing fibrillar collagen. Upon physiological activation (ADP, collagen) of patients’ platelets in vitro, there were no significant differences between the platelets of patients and healthy donors. However, the observed ex vivo size of platelet aggregates was significantly reduced in comparison with healthy donors and published data on patients with other thrombocytopenias. The observed number and activity (movement velocity) of granulocytes of patients was within normal values. However, significant morphological differences were observed for granulocytes of patients compared with granulocytes of healthy donors: there was an increased spreading of granulocytes, in particular, expressed in a large number of thin pseudopodia, as well as an increased curvature of the motion trajectories of granulocytes. Ex vivo observation of thrombus formation in patients with ANKRD26- associated thrombocytopenia, a significantly reduced thrombus size is observed with normal platelet activity and increased variability in the shape of granulocytes.

scholarly journals Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice

2019 ◽  
Vol 119 (08) ◽  
pp. 1321-1331
Author(s):  
Vaishali V. Inamdar ◽  
John C. Kostyak ◽  
Rachit Badolia ◽  
Carol A. Dangelmaier ◽  
Bhanu Kanth Manne ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Extracellular Region ◽  
C Terminus

Background and Objective CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. Methods and Results We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. Conclusion We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

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