The role of the T-lymphocyte in estrogen deficiency osteopenia.

Our laboratory has previously demonstrated that the T-lymphocyte is critical in the development of cyclosporin A-induced osteopenia in the rat model. A similar state of osteopenia is induced by estrogen depletion in the ovariectomized (OVX) rat, which is the animal model of postmenopausal bone loss. However, the role of the immune system, and particularly the T-lymphocyte, in estrogen deplete osteopenia has not been elucidated. We used the Rowett athymic nude rat as our model of T-lymphocyte deficiency. In this study, the experimental rats were divided into four groups as follows: (1) sham-operated Rowett heterozygous (rnu/+) euthymic rats (control group); (2) OVX Rowett heterozygous (rnu/+) euthymic rats; (3) sham-operated Rowett homozygous (rnu/rnu) athymic nude rats, which are T-lymphocyte deficient; and (4) ovariectomized Rowett homozygous (rnu/rnu) rats. Rats were weighed, and venous blood was taken in weeks 2, 4, and 6 for determination of serum osteocalcin. Serum 1,25-dihydroxyvitamin D (1,25(OH)2D) was determined on the day of sacrifice. Following sacrifice, histomorphometry was performed on double-labeled proximal tibial metaphyses. Flow cytometric analysis of splenic mononu-clear cell isolates stained for OX19-positive (CD5) T-lymphocytes was performed. T-lymphocyte analysis revealed significant reductions in both athymic nude groups, while OVX euthymic rats demonstrated a diminished number of T-cells relative to their sham-operated counterparts. Histomorphometric data indicated that both OVX groups exhibited a significant loss of trabecular volume, with associated increases in indices for bone formation and resorption, with resorption likely outstripping formation, resulting in osteopenia. Serum osteocalcin was significantly elevated in the ovariectomized euthymic group throughout the experimental period compared with the control group (p < 0.01); it was elevated in the ovariectomized athymic group on week 4 only (p < 0.01 vs. control). It appears that the T-lymphocyte may not be an essential component in the pathogenesis of estrogen deficiency osteopenia. The contribution of circulating T-lymphocytes as well as other T-lymphocyte-rich organs needs to be explored further.

Combined flurbiprofen and cyclosporin-A does not attenuate bone loss and exaggerates renal impairment.

Cyclosporine (CsA) is a potent immunosuppressant that has revolutionized the success of organ transplantation. Flurbiprofen (FB), a propionic acid derivative NSAID, has been demonstrated in vivo to reduce osteoclast numbers in normal rats. The aim of this experiment was to determine whether addition of FB to CsA-treated rats could prevent the bone changes associated with CsA therapy. Forty-eight 10-12-week-old male Sprague-Dawley rats were randomized to receive, daily for 28 days: (1) CsA vehicle p.o. plus FB vehicle sc; (2) CsA (15 mg/kg) p.o. plus FB vehicle sc, (3) CsA vehicle p.o. plus FB (1.5 mg/kg) sc; and (4) CsA (15 mg/kg) p.o. plus FB (1.5 mg/kg) sc. Rats were weighed and venous blood sampled at baseline, 14 days, and 28 days for determination of glucose, Ca+2, BUN, creatinine, PTH, osteocalcin, and 1,25(OH)2 vitamin D. Tibiae were removed following killing, after double labeling for histomorphometry. Body mass was significantly lower than control in all rats receiving CsA on days 14 and 28 while blood glucose was only elevated in the CsA alone group. Day 28 BUN and creatinine were significantly elevated in the CsA group and the combination of CsA and FB revealed an exacerbation of this trend. Vitamin D and osteocalcin were consistently increased in the CsA and CsA/FB groups. Bone histomorphometry showed evidence of trabecular osteopenia in CsA and CsA/FB groups. CsA alone resulted in elevated bone turnover. FB was unable to prevent the trabecular bone loss induced by CsA therapy. This experiment indicates no role for FB as a therapeutic option in CsA-induced bone disease at the given doses and duration of treatment by virtue of its lack of bone sparing ability and adverse renal effects when the two drugs are administered concurrently.

Short-term systemic insulin-like growth factor-1 is unable to prevent cyclosporin A-induced osteopenia in the rat.

Immunosuppression with cyclosporin A (CsA) is effective in a number of immune-mediated diseases and in preventing rejection following organ transplantation. We have repeatedly demonstrated that CsA in the rat model produces accelerated bone remodelling with net bone loss, best characterized in trabecular bone. IGF-I holds promise as a treatment for various osteopenic conditions. Although currently a subject of much controversy, various studies have suggested that in vivo it is anabolic to cortical as well as trabecular bone. The purpose of this study was, in part, to further characterize the effects of CsA and IGF-I on trabecular and cortical bone, and to see whether systemic IGF-I is able to modulate CsA's deleterious skeletal effects. Sixty 10 week-old, male, Sprague-Dawley rats were randomized to receive the following daily for 3 weeks: (1) CsA vehicle (veh) per os (po) + recombinant human (rh) IGF-1 veh subcutaneously (sc); (2) CsA 15 mg/kg po + rhIGF-I-veh; (3) CsA-veh + rhIGF-I 200 microg/kg sc; (4) CsA-veh + rhIGF-I 600 microg/kg sc; (5) CsA 15 mg/kg + rhIGF-I 200 microg/kg, and (6) CsA 15 mg/kg + rhIGF-I 600 microg/kg. Rats were weighed and venous blood was sampled serially for determination of glucose, ionized calcium (Ca2+), PTH, vitamin D, and osteocalcin. Following sacrifice on day 20, histomorphometry was performed on double calcein-labeled tibial metaphysis and diaphysis. All rats receiving CsA had elevated levels of blood glucose and osteocalcin by day 9 and vitamin D at day 20. PTH was similar in all groups, and Ca2+ was only raised in the CsA and CsA + IGF-I 200 microg/kg groups. Rats receiving IGF-I 200 microg/kg and IGF-I 600 microg/kg gained more weight than either vehicle- or CsA-treated animals, attesting to IGF-1's anabolic properties. CsA caused severe trabecular bone loss, not prevented by IGF-I; it even further increased the eroded surface. CsA and IGF-I had little effect on cortical bone volume or marrow area. IGF-I increased endocortical matrix synthesis, as evidenced by the increases in the percent endocortical osteoid perimeter, an effect negated by the addition of CsA. This experiment demonstrates that trabecular bone is more susceptible than cortical bone to the deleterious effects of CsA and indicates little role for IGF-1 in the pathophysiology or treatment of CsA-induced bone disease at the given doses and duration of treatment.

T lymphocytes play a critical role in the development of cyclosporin A-induced osteopenia.

The T lymphocyte suppressor, cyclosporin A, has been shown to cause high turnover osteoporosis. We postulated that cyclosporin A may exert its effects via the T cell rather than direct activity on bone. In this study we administered cyclosporin A (15 mg/kg.day by gavage) to 11 10-week-old Rowett athymic nude rats and to 12 age-matched immunocompetent Sprague-Dawley rats. Placebo was administered to control groups (n = 12 for both). After 28 days of treatment, the Sprague-Dawley rats displayed high turnover bone loss, but the nude rats were largely unaffected by the drug. Sprague-Dawley treated rats had less than half the percent trabecular area of their controls as measured at the secondary spongiosa of the proximal tibial metaphysis (P < 0.001; strain by treatment, P = 0.007). The same pattern was evident for trabecular number, separation, and thickness (strain by treatment, P = 0.034, P = 0.001, and P = 0.021, respectively). Only the Sprague-Dawley rats had an elevated percent eroded perimeter and an elevated bone area referent bone formation rate (strain by treatment, P = 0.002 and P = 0.0003, respectively). Mass, glucose, ionized calcium, PTH, osteocalcin, 1,25-dihydroxyvitamin D, and creatinine all responded similarly to cyclosporin A regardless of strain. T Lymphocytes thus appear to be a prerequisite for the development of cyclosporin A-induced osteopenia.

Bone mineral metabolism in T lymphocyte-deficient and -replete strains of rat.

The immune and skeletal systems are known to interact. We have repeatedly shown that in contrast to in vitro data, the administration of T lymphocyte immunosuppressants, such as cyclosporin A, leads to an increase in bone resorption and a high turnover osteopenia. The purpose of this study was to characterize the bone metabolism of the T lymphocyte deficient Rowett athymic homozygous (rnu/rnu) nude rat. We wished to determine whether these rats share the bone abnormalities of cyclosporin A-treated rats. Eleven 10-week-old Sprague-Dawley rats and 12 similarly aged nude rats were studied over a 4-week period. Metaphyseal cancellous bone histomorphometry was similar in the two groups of rats and only differed with regard to percentage eroded perimeter (lower in nude rats, p = 0.0008) and longitudinal growth rate (49% lower in nude rats, p < 0.001). The nude rats had less body mass (p < 0.001) but nevertheless gained the same percentage of their body weight over the study period. The athymic rats had lower levels of serum, 1,25-dihydroxyvitamin D (p < 0.014) and serum osteocalcin(p < 0.009), and at the age of 14 weeks the nude rats had lower concentrations of serum creatinine (p = 0.001) and blood ionized calcium (p = 0.0002), yet serum PTH was similar throughout. RNA isolated from the contralateral tibias revealed that the nude group had lower steady-state levels of osteocalcin mRNA despite similar rates of bone formation. In its entirety, the data suggest that T cell deficiency per se is not necessarily associated with high turnover osteopenia.

Rapamycin: a bone sparing immunosuppressant?

Immunosuppressant therpay is associated with osteoporosis both clinically, post-transplantation, and experimentally. In rats, cyclosporin A (CsA) and FK506 induce a state of high turnover rapid bone loss. After 14 days of administration in immunosuppressive doses, the more recently discovered immunosuppressant, rapamycin, resulted in no change of cancellous bone volume. A longer study over 28 days has now been carried out; contrasting the new drug with CsA and FK506. Sixty, 10-week-old Sprague-Dawley rats were randomly divided into five groups of 12 rats each. The first group served as an aging control. The remaining four groups received, by daily gavage, a combined vehicle placebo, CsA 15 mg/kg, FK506 5 mg/kg, and rapamycin 2.5 mg/kg, respectively. CsA- and FK506-treated rats, but not those treated with rapamycin, demonstrated high turnover osteoporosis with raised serum 1,25(OH)2D (p < 0.05) and elevated serum osteocalcin (p < 0.05). The trabecular bone area was decreased by 66% (p < 0.01) in the CsA group and 56% (p < 0.05) in the FK506-treated group compared with the control animals. The CsA- and the rapamycin-treated groups failed to gain weight and developed severe hyperglycemia (> 20 mmol/l, p < 0.001) by day 14 but which largely resolved by day 28. Unlike the groups treated with CsA and FK506, rapamycin-treated rats had no loss of trabecular bone volume but there was increased modeling and remodeling and a decreased longitudinal growth rate. Rapamycin may thus confer a distinct advantage over the established immunosuppressants in not reducing bone volume in the short term.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of medroxyprogesterone acetate on bone metabolism in the oophorectomized, tamoxifen-treated rat.

Tamoxifen (TAM) is used primarily in the management of breast cancer, and it also has bone-sparing effects similar to estrogen. In breast cancer patients TAM may have a potential role in the prevention and management of osteoporosis. TAM therapy is associated with uterine hyperplasia, and medroxyprogesterone acetate (MPA) added to the regimen provides protection against this. Due to the potential combined use of MPA and TAM in the clinical setting, this study was conducted to assess whether MPA acted synergistically, dampened, or enhanced the TAM effect on bone. Seventy-five female rats (60 oophorectomized; Ox), were randomized into five groups and received either TAM (0.1 mg/kg.day) and/or MPA (0.3 mg/kg.day) therapy over 28 days as follows: 1) sham; 2) Ox; 3) Ox plus TAM; 4) Ox plus MPA; and 5) Ox plus TAM plus MPA. Blood was sampled on days 0, 14, and 28 for measurement of ionized calcium, PTH, 1,25-dihydroxyvitamin D, osteocalcin, and insulin-like growth factor 1. TAM-treated rats showed a reduction in body weight serum osteocalcin, PTH, and insulin-like growth factor 1. Histomorphometric analysis of the proximal tibia showed less cancellous bone volume in Ox rats, and the effect was attenuated by TAM. MPA alone had no significant effect on cancellous bone volume. All the bone formation parameters evaluated (bone formation rate, mineral apposition rate, percent calcein-labeled surface, and number of osteoblasts) were higher in Ox rats compared with sham-operated rats and were lower in TAM-treated rats compared with Ox rats. These parameters were not changed by MPA, alone or in combination with TAM. The number of osteoclasts was higher in Ox rats compared with sham-operated rats and was reduced by TAM. MPA therapy alone or in combination with TAM did not affect number of osteoclasts. These results suggest that MPA neither dampened nor enhanced the effect of TAM on bone.

Amylin increases bone volume but cannot ameliorate diabetic osteopenia.

Amylin is normally secreted in a regulated fashion by the pancreatic beta-cells in parallel with insulin and has been reported to have bone-conserving properties. Type I diabetes mellitus results in a low-turnover osteopenia in the presence of decreased amylin, which is in contrast to type II diabetes where less bone loss, in the presence of high amylin levels, occurs. We investigated the effects of amylin on bone mineral metabolism in normal and diabetic (streptozotocin-induced) rats, in order to ascertain whether amylin would modify the streptozotocin-induced diabetic osteopenia. Ten-week-old male Sprague-Dawley rats were randomized as follows: group A (n = 18) received normal saline; group B (n = 18) received amylin; group C, diabetic rats (n = 23), received normal saline; and group D, diabetic rats (n = 23), received amylin. Amylin (100 pmol/100 g b.w.) was administered by a daily subcutaneous injection. Double calcein-labeled tibiae were removed for histomorphometric analysis followed sacrifice on day 19. Results showed no difference in blood ionized calcium between groups. Blood glucose remained above 600 mg/dl in the diabetic animals and was not affected by the administration of amylin. Serum osteocalcin, insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH), and 1,25 dihydroxyvitamin D [1,25(OH)2D] were significantly lower in the diabetic rats compared with control group A by day 19. Amylin produced higher levels of serum osteocalcin in group B on day 9 (P < 0.05) compared with controls but returned to control values (group A) by day 19; no such change occurred in the diabetic group.(ABSTRACT TRUNCATED AT 250 WORDS)

A new rapid and reproducible homologous immunoradiometric assay for amino-terminal parathyroid hormone in the rat.

Measurement of parathyroid hormone (PTH) in the rat is most often performed with competitive ligand radioimmunoassays (RIA) utilizing heterologous antibodies. We report here the validation of a newly developed homologous immunoradiometric assay (IRMA) for rat PTH. Two different goat antibodies to the amino-terminal sequence of rat PTH are utilized; one is immobilized onto plastic beads to capture the PTH molecules and the other is radiolabeled for detection. To test this new IRMA, 30 Sprague-Dawley rats were randomized into three treatment groups to receive by intraperitoneal injection: (1) saline 1 ml/kg (control); (2) calcium chloride 40 mg/kg (hypercalcemic); and (3) EDTA 300 mg/kg (hypocalcemic). Blood samples were taken at 0, 30, 60, 180, and 300 minutes after administration of the assigned treatment for measurement of ionized calcium (Ca2+) and serum PTH. Most of the variance in PTH levels was found to be due to changes in Ca2+ (r2 = 0.780, P < 0.0001). There was also a close temporal relationship between the two, with the highest levels of PTH occurring at the same measured time points as the lowest Ca2+, and vice versa. The measured detection limit of the IRMA was 3 pg/ml with intra- and interassay coefficients of variation of 1.74% and 3.07%, respectively. Serial dilutions with pooled rat serum, synthetic rat PTH-(1-34), and synthetic human PTH-(1-34) showed good parallelism with increased specificity for the pooled and synthetic PTH, despite a degree of crossreactivity with hPTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Azathioprine alone is bone sparing and does not alter cyclosporin A-induced osteopenia in the rat.

The immunosuppressant agent cyclosporin A (CsA) induces a high turnover osteopenic state, while the effect on bone of the antimetabolite azathioprine, a drug often used in conjunction with CsA in transplant patients, is less clear. This study was therefore designed to investigate the outcome of azathioprine administration, with reference to CsA, on bone mineral metabolism using the rat model. Four groups of 10-week-old male Sprague-Dawley rats (12 per group) were randomly allocated to receive by daily gavage for a 28-day period: (1) no treatment (control group); (2) azathioprine 1.5 mg/kg bw; (3) CsA 15 mg/kg bw; and (4) a combination of azathioprine and CsA, as described above. Rats were weighed and blood assayed serially for osteocalcin, ionized calcium, 1,25-dihydroxyvitamin D (1,25(OH)2VitD), and parathyroid hormone (PTH). Tibiae were removed following sacrifice on day 28 after double calcein labeling for histomorphometric analysis. Immunosuppressant groups were compared with nontreated control. We confirmed our previous findings that CsA induces a state of high turnover bone loss which is accompanied by a diminished gain in body weight (p < 0.01) and elevated serum osteocalcin (p < 0.001) and 1,25(OH)2VitD levels (p < 0.001). Azathioprine treatment alone did not alter ionized calcium, 1,25(OH)2VitD, or PTH levels. However, there was biochemical evidence of impaired osteoblastic activity as seen by decreased osteocalcin values on days 14 and 28 (p < 0.001). Azathioprine caused no loss of bone volume nor any deviation from the norm in mineral apposition rate, bone formation rate, or longitudinal bone growth. All three treatment groups showed an increased recruitment of osteoclasts to the bone surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Utilization of cyclosporine H to elucidate the possible mechanisms of cyclosporine A-induced osteopenia in the rat.

The immunosuppressive agent cyclosporine A (CsA) produces in vivo in the rat marked osteopenia and elevation of serum bone gla protein (BGP) that reflects the high bone turnover and increased circulating levels of 1,25-dihydroxyvitamin D [1,25(OH)2D]. Cyclosporine H (CsH), a D-N-MeVal11 analog of CsA, is not immunosuppressive, and in contrast to CsA, it neither binds to cyclophilin nor alters cytokine activity. This distinction between CsH and CsA provides a means of elucidating whether CsA exerts an effect on bone and 1,25(OH)2D via immune-mediated mechanisms. We therefore studied three groups of male Sprague-Dawley rats (300 to 350 g body weight [BW]) randomly divided to receive either cyclosporine vehicle, CsA, or CsH 15 mg/kg BW by daily gavage for 28 days. Blood samples were assayed for ionized calcium (Ca2+), serum BGP, serum parathyroid hormone (PTH), and 1,25(OH)2D by specific radioimmunoassay on days 0, 14, and 28. Histomorphometric evaluations were performed on the right tibia after tetracycline and calcein labeling and killing of the rats at the end of 28 days of treatment. In CsA-treated rats, serum BGP levels were significantly increased (155.2 +/- 30.7 ng/mL v 107.3 +/- 16.8 and 111.5 +/- 13.1 at day 28, P < .05) compared with control and CsH groups, respectively. Similarly, 1,25(OH)2D was significantly increased in CsA-treated rats versus control and CsH group (134.9 +/- 35.3 pg/mL v 70.2 +/- 16.6 and 69.8 +/- 20.6 [P < .05], respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin.

A potent inhibitor of platelet aggregation and cell adhesion was isolated from the venom of Bothrops atrox. This peptide, referred to as batroxostatin, was composed of 71 amino acids and showed a high degree of homology with other snake venom peptides including trigramin, albolabrin, elegantin and applagin: all 12 cysteines and the RGD sequence (standard one-letter amino acid codes) aligned in the same position. Compared on a molar basis, the anti-platelet aggregation activity of batroxostatin was about 1000-times higher than that of RGDS. In addition, batroxostatin was about 400-times more potent than GRGDS at inhibiting melanoma cell adhesion to fibronectin. Batroxostatin covalently attached to plastic promoted adhesion of melanoma cells. The anti-GP140 antibody, recognizing beta 1 integrins, completely inhibited adhesion of mouse melanoma cells to batroxostatin. This observation, in addition to the inhibitory effect of batroxostatin on the adhesion of chick fibroblasts to fibronectin, suggests that batroxostatin interacts with integrins from both the beta 1 and beta 3 subfamilies.

Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins. RGD-containing peptides from viper venoms.

Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein IIb/IIIa complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 microM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IIIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of beta-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of alpha granules and beta-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that disintegrins may be potential candidates for platelet protection during cardiopulmonary bypass.

Human platelet factor 4 and its C-terminal peptides: heparin binding and clearance from the circulation.

Human platelet factor 4 (PF4), a high affinity heparin binding protein, is released from stimulated platelets and stored at vascular sites, predominantly in liver, from where it can be brought back into circulation by heparin. We attempted to define structural requirements for PF4 binding to heparin and for the pattern of its clearance from the circulation. Intact PF4 bound strongly to heparin agarose and was eluted at 1.4 M NaCl, while reduced PF4 and PF4 C-terminal peptides PF4 (47-70) and PF4 (58-70) bound weakly and were eluted at 0.2-0.5 M NaCl. 125I-radiolabeled intact PF4, reduced PF4 and C-terminal PF4 peptides injected into rabbits were cleared from the circulation in a biphasic pattern with components having half-life time of 1-2 min and 20-140 min. Heparin eliminated the fast component of PF4 clearance, but it did not affect clearance of reduced PF4 or C-terminal PF4 peptides. In contrast to reduced PF4 and PF4 (47-70), intact PF4 that accumulated in the liver and spleen, was displaced by heparin into circulating blood. In conclusion, specific binding sites and native conformation of the molecule are critical for high affinity PF4 binding to insolubilized heparin and for a pattern of PF4 clearance from the circulation in the presence of heparin.

Elegantin and albolabrin purified peptides from viper venoms: homologies with the RGDS domain of fibrinogen and von Willebrand factor.

The RGD-containing peptides isolated from the venoms of the Viperidae constitute a new class of small cysteine-rich peptides of variable amino acid composition and biological activity (Huang, T.-F., et al. (1987) J. Biol. Chem. 262, 16157-16163; Gan, Z.R., et al. (1988) J. Biol. Chem 263, 19827-19832; Huang, T.-F., et al. (1989) Biochemistry 28, 661-668), which it is proposed by Gould et al. (unpublished data) that we call 'disintegrins'. These peptides bind to the glycoprotein IIb-IIIa receptor on the platelet surface and inhibit aggregation induced by ADP, thrombin, platelet-activating factor and collagen. These peptides are also potent inhibitors of cell adhesion to fibrinogen (Knudsen, K.M., et al. (1988) Exp. Cell Res. 179, 42-49). We report the isolation of two further RGD-peptides from the venoms of Trimeserusus elegans and Trimeserusus albolabris, purified to homogeneity with high yield by a novel, rapid reverse-phase HPLC method. The primary structures of these two peptides were determined to be single polypeptide chains of 73 amino acids. Albolabrin differed from trigramin by eight residues whilst elegantin differed by 22 residues. The molecular mass of albolabrin calculated on the basis of amino acid sequence was 7574 Da and the pI similarly calculated was 4.27. The molecular mass of elegantin was calculated to be 7806 Da and the theoretical pI to be 4.69. RGD is maintained in the same position (51-53 AA) and all 12 cysteines are identical. Our data suggest that the presence of RGD, the conserved secondary and tertiary structure, are essential for the expression of biological activity by these peptides. Both peptides inhibited ADP-induced platelet aggregation. Extended homologies around the RGDS sequences in human von Willebrand Factor and bovine fibrinogen were found with both peptides.

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.

Inhibition of platelet hemostatic plug formation by trigramin, a novel RGD-peptide.

Trigramin, a cysteine rich, RGD (Arg-Gly-Asp)-containing peptide from Trimeresurus gramineus snake venom (Mr 7,500) has been previously reported to inhibit fibrinogen binding to ADP-activated platelets and platelet aggregation (disassociation constant 10(-8) M). The present study demonstrates that the infusion of trigramin (17-212 micrograms/100 g body wt) significantly prolonged the bleeding time of severed mesenteric arteries in hamsters (anesthetized with 65 mg/kg pentobarbital), whereas the infusion of RGDS (Arg-Gly-Asp-Ser, 0.45-1.0 mg/100 g body wt) failed to increase the bleeding time in this model. The bleeding time immediately returned to normal after cessation of trigramin infusion. The pattern of the disappearance of 125I-labeled trigramin from the circulation fit a two-compartment model with the half-life for the fast component between 0.7 and 2.0 min and with the half-life for the slow component between 31 and 105 min. It appeared that the kidney and liver are major routes of elimination of trigramin from the circulation. The ability of trigramin to prolong bleeding time, as well as its rapid disappearance from the circulation, indicates that this peptide may be a useful compound to transiently prevent the ability of platelets to form thromboemboli without impairing their long-term hemostatic function.

Occurrence of platelet basic protein, a precursor of low affinity platelet factor 4 and beta-thromboglobulin, in human platelets and megakaryocytes.

beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.

Uptake and processing of human platelet factor 4 by hepatocytes.

We previously demonstrated rapid clearance of human platelet factor 4 (PF4) from rabbit and rat blood, its accumulation in the liver, and elimination of PF4 degradation products in urine. The purpose of the present experiments was to characterize interaction of PF4 with cultured rat hepatocytes. 125I-PF4 was taken up by hepatocytes reaching maximum at 180 min. The association of 125I-PF4 with hepatocytes was two times greater at 37 degrees C than at 4 degrees C. At 37 degrees C degradation of 125I-PF4 by hepatocytes was also observed as indicated by the increase of 125I-PF4 radioactivity soluble in 6% trichloroacetic acid. By contrast, no uptake of 125I-beta-thromboglobulin antigen was observed. Autoradiography demonstrated that short incubation (5-20 min) of 125I-PF4 with hepatocytes results in the association of 125I-radioactivity with cell membranes while after longer incubation (60 min) radioactivity was also localized in the endosomes. Heparin inhibited binding and uptake of 125I-PF4 radioactivity by hepatocytes. We propose that part of PF4 released in the circulating blood by activated platelets is bound to the surface of hepatocytes and that it is further processed by these cells.