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The underlying molecular basis for Bernard-Soulier Disease (BSD) is currently unknown. Platelets from patients with this autosomal recessive bleeding disorder have multiple abnormalities, including a markedly reduced von Willebrand factor-dependent adhesiveness due to a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex. In the present studies, we have used an intragenic restriction fragment length polymorphism (RFLP) for Taq I in the GPIb alpha gene to study linkage between this gene and the inheritance of BSD in a family with two affected siblings. Whereas the proband was heterozygous, showing both the 0.7 and 4.0 kb bands of this polymorphism (A/B), her affected brother was homozygous for the 0.7 kb band (A/A). Accordingly, these siblings did not inherit the same pair of GPIb alpha alleles from their parents. Additionally, one child of the proband was A/A, while the second studied child was A/B, with neither showing any evidence of BSD. No construct of heterozygosity or homozygosity for GPIb alpha alleles in this family is consistent with a model in which one or more defective GPIb alpha alleles could produce BSD. RFLP analysis with BamHI or HindIII showed entirely normal patterns in the patients, indicating the absence of any gross deletion of the GPIb alpha gene. GPIb alpha mRNA from patient platelets was reverse transcribed and subsequently amplified by the polymerase chain reaction, demonstrating the presence of GPIb alpha transcript. Furthermore, trace amounts of GPIb could be shown on the surface of patient platelets. Based on these results, a defect in the GPIb alpha gene is unlikely to be the cause of BSD in this family.

Previous studies performed in vitro have indicated that platelets from patients with platelet-type von Willebrand's disease (vWD) have receptors for von Willebrand factor (vWF) already exposed on their surfaces and that the addition of purified vWF or cryoprecipitate to patient platelet-rich plasma under stirring conditions is capable of inducing platelet aggregation and secretion. The present work reports the results of the transfusion of cryoprecipitate in a patient with platelet-type vWD. It is shown that, while factor VIII-related antigen and ristocetin cofactor activities maintain elevated levels for up to 12 hr following transfusion, the highest molecular weight vWF multimers decline rapidly. The platelet count also declines, followed in turn by a rise in the plasma level of platelet factor 4. Shortening of the bleeding time occurs only very transiently. The results of this study provide direct evidence that, in patients with platelet-type vWD, an abnormal interaction of their platelets with plasma vWF occurs in vivo, resulting in the absence of high molecular weight vWF multimers, low platelet counts, and impaired hemostasis that are characteristic of this disease.

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

Megakaryocytes share a number of structural and chemical properties with their progeny, blood platelets. With the availability of highly purified preparations of megakaryocytes isolated from guinea pig bone marrow, it is now also possible to study functional aspects of these cells. The present work reports the first study of the release of endogenously stored materials in megakaryocytes. Guinea pig megakaryocytes isolated to 75%-90% purity were exposed to thrombin or calcium ionophore (A23187) and the release of ATP was continuously monitored with the luciferin-luciferase reaction. Both maximal extent and initial rate of release were studied. Thrombin-induced release was half-maximal at thrombin concentrations of 0.2–0.5 NIH U/ml. At 4 U/ml thrombin, maximal release was 538 +/- 147 nmole ATP/10(9) megakaryocytes. A23187 induced half-maximal responses at concentrations of 7–8 microM. ATP release by ionophore showed a nearly absolute requirement for extracellular calcium, with release by thrombin showing only a partial calcium dependence. Following overnight culture, the response to thrombin was unchanged, whereas ATP release in response to ionophore was consistently increased (p less than 0.01). By comparison of maximally releasable ATP with total cellular ATP content, the storage pool of ATP in megakaryocytes was determined to comprise only 2%-6% of total megakaryocyte ATP, in contrast to an ATP storage pool of 20%-30% in guinea pig platelets. This difference may reflect further entry of ATP into the storage pool compartment or an enhanced ability of the cell to recognize and respond fully to platelet stimuli as the megakaryocyte reaches full maturity.

The primary sequences of the three individual glycoprotein (GP) chains, GPIb alpha, GPIb beta, and GPIX, comprising the normal platelet GPIb/IX receptor for von Willebrand factor (vWF) have recently been determined, opening the possibility for characterization of disorders of this receptor at the molecular level. The presence of a leucine tandem repeat in each of these chains is of particular interest, because such repeats may be involved in associations between polypeptide segments. We now report an autosomal dominant variant of Bernard-Soulier disease associated with the heterozygous substitution of phenylalanine for a highly conserved leucine residue within the GPIb alpha leucine tandem repeat. Affected individuals experienced a moderate bleeding tendency, thrombocytopenia, and an increased mean platelet volume. Platelet aggregation was decreased only in response to ristocetin or to asialo- vWF. The kd for 125I-vWF binding to patients platelets was significantly increased over control values at 0.5 mg/mL ristocetin, but was normal at 1.0 or 1.5 mg/mL ristocetin. While sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an essentially normal complement of all components of the GPIb/IX complex, a minor amount of a putative proteolytic fragment was identified that migrated faster than GPIb and was immunoreactive with polyclonal anti-GPIb alpha antibody, but not with a monoclonal antibody directed against the 45-Kd amino-terminal region of GPIb alpha. However, because the great majority of patient GPIb alpha comigrates with normal GPIb alpha, the major functional abnormalities of the patient platelets are most likely a consequence of the altered structure of the nonproteolyzed protein. Full concordance within the studied family between phenotypic expression and a heterozygous single nucleotide substitution in genomic DNA coding for a phenylalanine in place of the wild-type leucine at residue 57 of the mature GPIb alpha, absence of this substitution in 266 alleles from the normal population, and the lack of any other abnormality of patient DNA throughout the entire coding sequence for GPIb alpha provide strong support that this substitution may constitute a pathologic point mutation responsible for the observed phenotypic abnormalities. While the roles that leucine tandem repeats may normally play within the GPIb/IX complex are not yet known, the perturbation of such a repeat in GPIb alpha may impair interaction with other components of the complex and/or with the binding of vWF.

Previous studies in the guinea pig model system have established a close structural homology between human and guinea pig glycoproteins Ib (GPIb) and IIb/IIIa (GPIIb/IIIa). Moreover, the murine monoclonal antibody (MoAb) PG-1, which recognizes GPIb in guinea pig platelets and megakaryocytes, exerted full inhibition on von Willebrand factor (vWF)- dependent platelet agglutination without inhibiting aggregation induced by ADP, collagen, or thrombin. The present research extends this animal model system to study of the effects on hemostatic function following the in vivo injection of MoAb PG-1 or its F(ab')2 fragments. A hind limb template bleeding time methodology was developed for use in guinea pigs. Normal bleeding time was determined to be 2.7 +/- 0.5 minutes (mean +/- SD), with an observed range of two to four minutes. Platelet counts in these same animals were 501 +/- 82 x 10(3)/microL. After intraperitoneal (IP) injection of busulfan, guinea pigs became increasingly thrombocytopenic. As long as the platelet count remained above approximately 150 x 10(3)/microL, the bleeding time was not more than five minutes; however, further decrease in the platelet count was accompanied by more marked prolongations of the bleeding time. For 14 to 72 hours after IP injection of 1.3 mg/kg intact PG-1 MoAb, a hemorrhagic state was produced with a bleeding time greater than 20 minutes. The platelet count concurrently decreased to approximately 50% of its baseline value but could not be further decreased either by raising the initial PG-1 dosage tenfold or by administering a second, equal dose 24 hours after the initial injection. This finding may reflect a heterogeneity of circulating platelets with respect to GPIb, to Fc receptors, or to an interaction between them. After IP injection of 0.63 to 2.5 mg/kg PG-1 F(ab')2 fragment, platelet counts did not decrease more than 21% below baseline levels in a 72-hour period, and bleeding times never increased by more than one minute over baseline values. Nevertheless, platelets obtained from animals 24 hours after injection of 2.5 mg/kg PG-1 F(ab')2 showed full inhibition of agglutination induced by ristocetin. The response of these platelets to aggregation by asialo-vWF was also severely inhibited as compared with control platelets. PG-1 F(ab')2 produced no effect on aggregation induced by ADP. These studies show that virtually complete functional block of the vWF receptor by F(ab')2 fragments of the anti-GPIb MoAb PG- 1 is not sufficient to produce a hemorrhagic state in the guinea pig animal model system.

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.

An autosomally transmitted bleeding diathesis sharing some, but not all, features previously described in von Willebrand's disease (vWd) was studied in five patients representing three generations of a single family. Bleeding times in the upper normal range in conjunction with low-normal platelet counts, normal factor VIII coagulant activity and VIII-related antigen, decreased VIII-ristocetin cofactor activity, selective decrease of the higher molecule weight factor VIII/von Willebrand factor (VIII/vWF) multimers, and increased ristocetin- induced platelet agglutination at low ristocetin concentrations were characteristic. Binding of patient VIII/vWF to washed normal platelets was within normal limits, whereas binding of normal VIII/vWF to patient platelets was significantly increased (p less than 0.001 at 0.6 mg/ml ristocetin). This disorder accordingly appears to involve an intrinsic platelet abnormality affecting platelet-VIII/vWF interactions. It is proposed that the concept of vWD be broadened to include patients with this abnormality, which may appropriately be called “Platelet-type von Willebrand's disease.”

Early thrombosis of artificial microvascular grafts (AMG, grafts < or = 2 mm internal diameter) prevents their reliable clinical use. The present studies were undertaken to examine the effect of hirudin, a thrombin-specific inhibitor, and of the F(ab')2 fragment of PG-1, a monoclonal antibody (MoAb) directed against guinea pig platelet membrane glycoprotein Ib (GPIb), on AMG patency in an animal model. One- centimeter long segments of expanded polytetrafluoroethylene (ePTFE), 0.88 mm internal diameter, were serially implanted as interposition grafts in the guinea pig femoral arterial systems bilaterally. A control group was treated with 0.5 mL saline intravenously (IV) 30 minutes before limb 1 and limb 2 graft implantation. Three experimental groups were treated with 0.5 mL saline IV before limb 1 graft implantation as an animal control and with either 0.5 mL saline containing 1.25 mg/kg IV PG-1 F(ab')2, (which inhibits ristocetin- induced platelet agglutination and von Willebrand factor binding), hirudin 1 mg/kg IV, or a combination of both agents before limb 2 graft implantation. GPIb inhibition, thrombin inhibition, and the combination resulted in a significant prolongation of AMG patency (P < .005). Whereas thrombin inhibition with hirudin prolonged AMG patency similar to that observed with GPIb inhibition, the combination of GPIb and thrombin inhibition provided the overall longest prolongation of AMG patency. These results indicate that both platelet membrane GPIb and thrombin play a role in AMG thrombosis.

Analogues of the neurotransmitter substance P (SP) can interact with neuropeptide receptors, and are reported to inhibit growth of small cell lung cancer cell lines (SCLC CLs). We found [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P (D-Phe5SP) significantly inhibited DNA synthesis by 10/10 human tumour CLs; six SCLC, one N-SCLC (squamous), two ovarian and one squamous cervical carcinoma, with inhibition to 50% control levels (IC50) of 20-50 microM. There was dose dependent inhibition of colony forming efficiency (CFE) in 3/3 SCLC and 1/1 N-SCLC CL, IC50s of 0.5-6.5 microM in 5% serum. Exposure of SCLC CL HC12 to 100 microM D-Phe5SP for 1-4 h caused a progressive fall in viable cell number; surviving cells, grown in the absence of peptide, showed a decreased growth rate. During 1 week's exposure of two SCLC CLs to 20 microM D-Ph5SP, growth was slower than control cultures, while 50-100 microM completely inhibited growth. These inhibitory effects were partially reversed by increasing serum concentration from 5 to 20%, but not by SP, vasopressin, bombesin or insulin-like growth factor 1. There was some inhibition of CFE by 3/3 normal human bone marrows, IC50s of 30-80 microM, compared with 8 microM for HC12 in 20% FCS. Therefore D-Phe5SP appears to have more potent antiproliferative effects in tumour cells than normal cells, suggesting a role for this analogue in tumour treatment.