Molecular level studies on platelets deficient in collagen-induced aggregation provide evidence for identifying possible platelet collagen receptors. We investigated platelets from a patient with mild bleeding time prolongation, but otherwise normal coagulation data. Her platelets lacked collagen-induced aggregation and adhesion, but retained normal aggregation and release by other agonists. Labeling her platelets with 125I or 3H and analysis by SDS-PAGE/autoradiography showed normal levels of glycoproteins Ia, Ib, Ila, IIb, IIIa, and IV. However, there were significantly decreased incorporations of both radioactivities into a 61-kD membrane glycoprotein (GP), which was identified as GPVI from its mobility on unreduced-reduced, two-dimensional SDS-PAGE. Sugiyama et al. ( . Blood. 69: 1712 reported that the serum from an idiopathic thrombocytopenic purpura (ITP) patient contained an antibody against a 62-kD platelet protein. Our patient's platelets lacked the antigen for the ITP patient's antibody, demonstrating that the ITP serum contains a specific antibody against GPVI. The patient's parents' platelets contained -50% the normal amount of GPVI, but still had normal collagen-induced aggregation and adhesion. The patient's platelets did not bind to types I and III collagen fibrils. Our results suggest that GPVI functions as a collagen receptor.

The gene coding for plasminogen has been compared with several abnormal genes from Japanese patients by the polymerase chain reaction and DNA sequence analysis. Two types of abnormal genes coding for plasminogen were identified in these patients. In the type I mutation, a guanosine in _GCT coding for Ala-601 near the active-site histidine was replaced by an adenosine resulting in ACT coding for threonine. This mutation was also shown by the loss ofa cleavage site for Fnu4Hl endonuclease, a restriction enzyme that recognizes -GCTGC but not ACTGC. In the type II mutation, a guanosine in IiTC coding for Val-355 was replaced by a thymidine resulting in TTC coding for phenylalanine. This change was readily shown by digestion with Ava II endonuclease, a rehtriction enzyme that recognizes GfiTCC and not GTTCC. The type I mutation has been found to be identical to a pinogen variant identified in Japanese patients by amino acid sequence analysis and also detected by isoelectric focusing, whereas the type II mutation is a unique amino acid substitution in the connecting region between the third and fourth kringles in plasminogen. DNA sequence analysis also revealed that the abnormal genes carry several silent nucleotide substitutions located primarily within introns and 5' and 3' flanking regions.

Platelet adhesion to the exposed surface of the extracellular matrix in flowing blood is the first and critical reaction for in vivo thrombus formation. However, the mechanism of this in vivo platelet adhesion has yet to be studied extensively. One of the reasons for this is the lack of a practical assay method for assessing platelet adhesion under flow conditions. We have devised an assay method (the fluorescent adhesion assay) that is based on the technique originally reported by Hubbell and McIntire (Biomaterials 7:354, 1986) with some modifications to make it more amenable for assaying small samples and have developed an analysis method to quantify the extent of platelet adhesion and aggregation from fluorescence images by using a computer-assisted image analysis system. In our assay, platelet adhesion, expressed as the percentage of the area covered by adhered platelets, was found to increase biphasically as a function of time. In the first phase, platelets interacted with the coated collagen, transiently stopping on the surface; we called this reaction the temporary arrest. In the second phase, platelets adhered much more rapidly and permanently on the surface, and this adhesion was dependent on the shear rate; platelets formed aggregates in this phase. We used our assay to analyze the effects of platelet aggregation inhibitors on platelet adhesion. All three examined inhibitors, EDTA (10 mmol/L), antiglycoprotein (GP) IIb/IIIa, and GRGDS peptide (1 mmol/L), inhibited the second phase adhesion in flowing blood. Furthermore, GPVI-deficient platelets also showed defective second-phase adhesion under the same conditions. These results suggested that GPIIb/IIIa activation and GPVI contribute to the reaction inducing the second phase. The second-phase adhesion has been extensively investigated, and the consensus is that this reaction is mainly attributable to the platelet-platelet interaction. In this report, we were able to detect an earlier reaction, the temporary arrest. This temporary arrest would reflect the fast and weak interaction between platelet GPIb/IX and collagen-von Willebrand factor complexes on the collagen-coated surface.

The HLA types of peripheral blood lymphocytes (PBL) of 36 adult T-cell leukemia/lymphoma (ATLL) patients were examined and compared with those of 45 healthy relatives of these patients, and with those of 10 non-ATLL families including 80 healthy members. Thirty-one percent of ATLL patients showed either a gain or a loss of HLA antigens determined by the presence of alien HLA antigens or the absence of inherent HLA antigens deduced from familial haplotype analysis. The antigen specificity of HLA gained or lost was variable and differed from case to case among ATLL patients. Although the gain of HLA was detected only in ATLL patients, the loss of HLA was found both in ATLL patients and in asymptomatic healthy relatives. The rate of HLA loss in ATLL patients (8.4%) and their relatives (17.8%) was much higher than in relatives of non-ATLL patients (1.1%). The HLA gain and loss revealed in the PBL of ATLL patients were confirmed by altered HLA phenotypes in the cloned T and B cells established from ATLL patients. Our results suggest that latently infecting HTLV-I may induce altered HLA phenotypes in T and B cells, primarily with loss of HLA antigens in a population of asymptomatic virus carriers, and secondarily with a gain of HLA antigens after the development of ATLL.

A 25-year-old male physician with acute hepatitis C after needle-stick injury was treated with combination therapy including twice-a-day interferon-beta (IFN-beta) and standard interferon-alpha (IFN-alpha). The infecting strain was of genotype 1b. Pretreatment hepatitis C virus (HCV) RNA levels were high. Because severe paresthesias occurred with initial daily administration of 5 million units (MU) of lymphoblastoid IFN-alpha, the dose was reduced to 3 to 6 MU of IFN-alpha2b three times a week. However, HCV RNA was not cleared from serum after 20 weeks of standard IFN-alpha2b treatment. A 4-week course with IFN-beta, at the dosage of 3 MU twice daily i.v. drip, was then started and followed by an 18-week course with IFN-alpha2b, 6 MU thrice weekly. After IFN-beta treatment, HCV RNA was cleared from serum without severe adverse effects, including paresthesias. Total amounts of IFN administered were 20 MU of lymphoblastoid IFN-alpha, 648 MU of IFN-alpha2b, and 252 MU of IFN-beta. Complete response and avoidance of chronic HCV infection were achieved. Thus, combination therapy with twice-a-day IFN-beta and standard IFN-alpha was effective in treating an acute hepatitis C patient with a high viral load and sensitivity to adverse effects of high-dose IFN-alpha.