guanosine-triphosphate and Hemorrhage

The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.

Topics: Adenosine Diphosphate; Blood Coagulation Disorders, Inherited; Blood Platelets; Cell Line; Female; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Hemorrhage; Heterozygote; Homozygote; Humans; Male; Megakaryocytes; Membrane Proteins; Mutation; Neoplasm Proteins; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Shelterin Complex; Telomere-Binding Proteins

Rats subjected to polytrauma and hemorrhage develop a coagulopathy that is similar to acute coagulopathy of trauma in humans, and is associated with a rise in prothrombin time and a fall in clot strength. Because platelet aggregation accounts for a major proportion of clot strength, we set out to characterize the effects of polytrauma on platelet function.. Sprague-Dawley rats were anesthetized with isoflurane. Polytrauma included laparotomy and damage to 10 cm of the small intestines, right and medial liver lobes, right leg skeletal muscle, femur fracture, and hemorrhage (40% of blood volume). No resuscitation was given. Blood samples were taken before and after trauma for the measurement of impedance electrode aggregometry, and intracellular levels of cyclic adenosine and guanosine monophosphate (cAMP, cGMP), inositol trisphosphate (IP3), and adenosine and guanosine triphosphates (ATP, GTP).. Polytrauma significantly increased the response of collagen (24%) and thrombin (12%) to stimulate platelet aggregation. However, aggregation to adenosine diphosphate (ADP) or arachidonic acid (AA) was significantly decreased at 2 (52% and 46%, respectively) and 4 h (45% and 39%). Polytrauma and hemorrhage also led to a significant early rise in cAMP (101 ± 11 to 202 ± 29 pg/mL per 1,000 platelets), mirrored by a decrease in cGMP (7.8 ± 0.9 to 0.6 ± 0.5). In addition, there was a late fall in ATP (8.1 ± 0.7 to 2.2 ± 0.6 ng/mL per 1,000 platelets) and GTP (1.5 ± 0.2 to 0.3 ± 0.1). IP3 rose initially, and then fell back to baseline.. Polytrauma and hemorrhage led to a deficit in the platelet aggregation response to ADP and AA after trauma, likely due to the early rise in cAMP, and a later fall in energy substrates, and may explain the decrease in clot strength and impaired hemostasis observed after severe trauma.

Topics: Adenosine Triphosphate; Animals; Blood Coagulation Disorders; Disease Models, Animal; Guanosine Triphosphate; Hemorrhage; Male; Multiple Trauma; Platelet Aggregation; Rats; Rats, Sprague-Dawley