A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize a-thrombin-and peptide ligand-induced activation responses using two different antibodies: anti-TR"52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR'" generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both athrombin (10 nM) and peptide ligand (20 MM) were studied using fura 2-loaded cells and microspectrofluorimetry.

A functional thrombin receptor (TR) structurally related to other members of the seven-transmembrane receptor family has been isolated from diverse cellular types intimately involved in the regulation of the thrombotic response. This receptor recapitulates many of the previously identified sequelae of thrombin-mediated cell activation phenomenon, and requires proteolytic cleavage for downstream effector- response coupling events. Using two complementary approaches, we have now completed the chromosomal assignment of the human thrombin receptor gene. Discordancy analysis of polymerase chain reaction products from a human-rodent hybrid cell mapping panel assigned the sequence to human chromosome 5 with no observed discordancies. Cytogenetic localization using fluorescence in situ hybridization on human metaphase chromosomes specifically localized the human TR gene to region q13 of chromosome 5, confirming its presence as a single-locus gene in the human genome. The chromosomal localization of the human TR gene is at or contiguous with the proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anemia).

von Willebrand disease (vWD), one of the most common bleeding disorders in humans, is manifested as a quantitative or qualitative defect in von Willebrand factor (vWF), an adhesive glycoprotein (GP) with critical hemostatic functions. Except for the rare severely affected patient with a gene deletion as etiology of the disease, the molecular basis for vWD is not known. We studied the molecular basis for vWD in a breeding colony of pigs with a disease closely resembling the human disorder. The porcine vWF gene is similar in size and complexity to its human counterpart, and no gross gene deletion or rearrangement was evident as the pathogenesis of porcine vWD. A restriction fragment- length polymorphism (RFLP) within the porcine vWF gene was identified with the restriction endonuclease HindIII, and 22/35 members of the pedigree were analyzed for the polymorphic site. Linkage between the vWF locus and the vWD phenotype was established with a calculated LOD score of 5.3 (1/200,000 probability by chance alone), with no crossovers identified. These findings indicate that porcine vWD is due to a molecular defect within (or near) the vWF locus, most likely representing a point mutation or small insertion/deletion within the vWF gene.

A functional thrombin receptor (TR) structurally related to other members of the seven-transmembrane receptor family has been isolated from diverse cellular types intimately involved in the regulation of the thrombotic response. This receptor recapitulates many of the previously identified sequelae of thrombin-mediated cell activation phenomenon, and requires proteolytic cleavage for downstream effector- response coupling events. Using two complementary approaches, we have now completed the chromosomal assignment of the human thrombin receptor gene. Discordancy analysis of polymerase chain reaction products from a human-rodent hybrid cell mapping panel assigned the sequence to human chromosome 5 with no observed discordancies. Cytogenetic localization using fluorescence in situ hybridization on human metaphase chromosomes specifically localized the human TR gene to region q13 of chromosome 5, confirming its presence as a single-locus gene in the human genome. The chromosomal localization of the human TR gene is at or contiguous with the proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anemia).