Abstract-Extracellular matrix (ECM) regulates vascular smooth muscle cell proliferation. The role of ECM in myocardial growth is unexplored. We sought to determine whether human fetal ventricular myocytes (HFVMs) produce ECM and whether synthesis and attachment to ECM are necessary for their epidermal growth factor (EGF)-dependent and -independent proliferation. Cultured HFVMs proliferate in the presence but not absence of serum and EGF, as determined by increase in cell number and [ 3 H]thymidine and [ 14 C]leucine incorporation (measures of DNA and protein synthesis, respectively). Using a cyanogen bromide digestion technique to measure collagen and elastin and using affinity chromatography for fibronectin, we found that HFVMs synthesized collagen and fibronectin but not elastin. HFVMs grown on exogenous ECM (including fibronectin and type I collagen and laminin) demonstrated no change in proliferation or DNA and protein synthesis with or without EGF. However, inhibition of collagen synthesis using cis-4-hydroxyproline resulted in a decrease in EGF-related HFVM proliferation and DNA and protein synthesis, which was reversed by exposure to L-proline but not by growth on type I collagen. Use of ␤ 1 but not ␤ 3 integrin antibody to inhibit cell interaction with ECM resulted in a decrease in HFVM proliferation and DNA and protein synthesis in response to EGF. Furthermore, EGF-dependent proliferation was enhanced by ␣ 1 ␤ 1 and ␣ 5 ␤ 1 antibodies that act as functional ligands, but not ␣ 3 ␤ 1 , the only ␤ 1 subtype expressed in adult myocytes. In conclusion, proliferating HFVMs synthesize collagen and fibronectin. The proliferative response of HFVMs to EGF requires the synthesis of collagen as well as attachment to specific ␣/␤ 1 integrin heterodimers. (Circ Res. 2000;87:508-515.)

SummaryLysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.

ing men produced more thromboxane B2 (3.99±0.76 ng/ml) than nonsmoking women (2.13±0.24 ng/ml). Female smokers produced more thromboxane B2 (5.01±0.97 ng/ml) than nonsmoking women. Twenty-four hours after a single dose of 600 mg aspirin, in vitro production of thromboxane B2 in response to collagen fell by 95%, whereas in vivo production of thromboxane B2 and 6-keto-PGF,a in bleeding time blood fell by 87% and 66%, respectively. Subjects with the lowest absolute levels of thromboxane B2 24 hours after aspirin were also those with the longest postaspirin bleeding times. Recovery of 6-keto-PGFla production was faster than recovery of thromboxane B2 production, but 6-keto-PGFI, production for most subjects was still below basal 72 hours after aspirin. The influence of two different doses of long-term aspirin (80 mg every other day and 325 mg daily) on the in vivo production of thromboxane B2 and 6-keto-PGFl. was studied in normals and diabetics. After 14 days of 80 mg aspirin every other day, thromboxane B2 and 6-keto-PGFIa production were both substantially inhibited (93% and 78%, respectively). After 14 days of 325 mg aspirin daily, thromboxane B2 production was similarly substantially inhibited (93%), whereas 6-keto-PGFIa was significantly less affected (only 45% inhibition). Study of a second group of five normal subjects confirmed that 6-keto-PGFl. production was significantly inhibited 24 hours after the first dose of 325 mg aspirin but was not significantly less than basal after 14 days of 325 mg aspirin. The results suggest that 325 mg aspirin daily is more antithrombotic compared with 80 mg every other day due to the superior preservation of prostacyclin production. (Circulation 1989;79:29-38) T hromboxane A2, a potent vasoconstrictor and platelet-aggregating agent produced by platelets, and prostacyclin, a potent vasodilator and platelet inhibitor produced by vascular tissue, are believed to exert critical modulatory roles in vivo.1,2 Variations in the production of these comFrom the

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.

Exercise at a heart rate corresponding to 30% VO2max for 15 min was associated with an increase in the volume of bleeding time blood from a mean of 133 microliters before exercise to a mean of 218 microliters during and immediately after the exercise. There was similarly an increase in thromboxane B2 production from 6.40 nmol.l-1 before to 11.50 nmol.l-1. Most subjects also showed an increase in the length of the bleeding time and in the production of bleeding time 6-keto-PGF1 alpha. The extent of increase in the bleeding time and in production of 6-keto-PGF1 alpha was quite variable, with subjects showing the largest increases in bleeding time also demonstrating the greatest increases in 6-keto-PGF1 alpha (r = 0.76, P = 0.004). The ingestion of aspirin before exercise markedly inhibited basal bleeding time thromboxane B2 production and blocked the exercise-associated increments in thromboxane B2 and 6-keto-PGF1 alpha production. While the aspirin itself increased the length of the bleeding time, there was not any further increase associated with exercise. In contrast to the effects of acute short-term exercise, long-distance running was associated with a significant decrease in bleeding time, but no change in bleeding time blood volume, bleeding time thromboxane B2, or bleeding time 6-keto-PGF1 alpha. The results show that acute low-level exercise can be associated with significant changes in the volume of blood oozing from a bleeding time incision and in the amount of thromboxane production stimulated at the incisional site. Following exhaustive exercise of long duration, the above changes are no longer seen.

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3-8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (less than 30% inhibition) and MLC (less than 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.

No abstract

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.