Calcimimetic compound NPS R-568 stimulates calcitonin secretion but selectively targets parathyroid gland Ca(2+) receptor in rats.

N-(3-[2-Chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS R-568) is an orally active compound that activates Ca(2+) receptors on parathyroid cells and rapidly suppresses plasma levels of parathyroid hormone (PTH) and Ca(2+) (ED(50), 1 and 10 mg/kg, respectively). We now show that increased calcitonin secretion contributes to NPS R-568-induced hypocalcemia. In parathyroidectomized thyroid-intact rats in which normocalcemia was restored by PTH infusion, NPS R-568 rapidly reduced plasma Ca(2+) levels, indicating that decreased PTH secretion was not solely responsible for the hypocalcemia seen in normal animals. NPS R-568 decreased plasma Ca(2+) levels in thyroidectomized parathyroid-intact rats, but the rate of onset of hypocalcemia was slower than in controls. In contrast, NPS R-568 had no effect on plasma Ca(2+) levels in PTH-infused, thyroparathyroidectomized rats, providing evidence that increased calcitonin secretion caused the hypocalcemia in PTH-infused parathyroidectomized rats. NPS R-568 rapidly increased plasma calcitonin levels to a peak at 10 to 20 min after oral dosing (ED(50) 40 mg/kg). NPS R-568 did not affect the rate of disappearance of (45)Ca from blood, indicating that hypocalcemia resulted from decreased influx of Ca(2+) into the circulation and not from increased efflux. This suggests that NPS R-568-induced hypocalcemia resulted solely from reduced efflux of Ca(2+) from bone after increased calcitonin and reduced PTH secretion. Thus, NPS R-568 causes hypocalcemia by activating Ca(2+) receptors on C cells and parathyroid cells; however, NPS R-568 is about 40 times more potent in reducing PTH levels than in increasing calcitonin levels.     

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.     

Positive correlation of serum bio-intact PTH(1-84) but not intact PTH with parathyroid gland size in hemodialysis patients.

To evaluate the usefulness of newly-developed bio-intact parathyroid hormone (Bio-PTH) assay, which measures exclusively intact PTH(1-84) molecule, serum PTH level determined by Bio-PTH assay, in comparison with second-generation intact PTH (I-PTH) assay, was examined for its correlation with parathyroid gland size. Serum PTH was determined in 46 male HD patients, together with bone formation markers bone alkaline phosphatase, intact osteocalcin, N-terminal propeptide of type I collagen, and bone resorption markers deoxypyridinoline, pyridinoline, beta-crossLaps. Maximal diameter of parathyroid gland was determined with ultrasonography as the parathyroid gland size. Serum Ca and Pi correlated significantly with parathyroid gland size rationalizing our method to define parathyroid gland size. Serum Bio-PTH was correlated significantly in a positive manner with parathyroid gland size (R = 0.308, P = 0.0474), whereas serum I-PTH did not. Furthermore, parathyroid gland size did not exhibit a significant correlation with any of bone formation markers or bone resorption markers. The lack of correlation between bone markers and parathyroid gland size in HD patients may be explained by the occurrence of refractoriness of bone to PTH. In conclusion, serum Bio-PTH assay could provide a better assay than I-PTH assay to estimate parathyroid function in HD patients, due mainly to its exclusive correlation with parathyroid gland size.     

Human transforming growth factor-alpha stimulates bone resorption in vitro.

Tumor-derived transforming growth factors (TGF) have been proposed as possible mediators of hypercalcemia in malignancy. We have studied the action of recombinant human TGF-alpha in cultured bone cells and in bone explant cultures. In clonal UMR-106 rat osteosarcoma cells, TGF-alpha and epidermal growth factor (EGF) were equipotent in binding to the EGF receptor. TGF-alpha and EGF both stimulated resorption of neonatal mouse calvaria, and maximal responses were obtained with 10 ng/ml of TGF-alpha after 72 h in culture. The effects of both TGF-alpha and EGF in calvaria, but not those of parathyroid hormone, were inhibited by 5 X 10(-7) M indomethacin. Fetal rat limb bone cultures were less sensitive to TGF-alpha than neonatal mouse calvaria, with a concentration of 30 ng/ml being required to stimulate resorption in this system. The bone-resorbing activity of TGF-alpha in fetal rat bones was inhibited by 10 ng/ml calcitonin but not by 5 X 10(-7) M indomethacin. EGF at concentrations up to 300 ng/ml did not stimulate resorption of the limb bones at time periods up to 66 h. The results indicate that human TGF-alpha is a potent bone-resorbing agent, and support the concept that this growth factor exhibits some effects distinct from those of EGF. TGF-alpha could play an etiologic role in the hypercalcemia of malignancy.     

Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol.

The present study determined the effects of 1,25-dihydroxycholecalciferol on serum immunoactive parathyroid hormone and on parathyroid hormone secretion in vitro. Rats injected i.p. with 1,25-dihydroxycholecalciferol, 130 pmol (2 U)/140 g body wt, which is probably a physiologic dose, had a significant 43% decrease in serum immunoreactive parathyroid hormone at 4 h. In addition, this dose of 1,25-dihydroxycholecalciferol inhibited the serum immunoreactive parathyroid hormone response to hypocalcemia induced by phosphate injection. Because the increment in serum immunoreactive parathyroid hormone was less but the decrement in serum calcium more in phosphate plus 1,25-dihydroxycholecalciferol-treated than in phosphate plus vehicle-treated rats, the impaired serum immunoreactive parathyroid hormone response to 1,25-dihydroxycholecalciferol could not be attributed to the change in serum calcium. In studies of parathyroid hormone secretion from bovine parathyroid tissue in vitro, the concentration of 1,25-dihydroxycholecalciferol used for most experiments was 1nM, which is in the range found in rat serum. 1,25-Dihydroxycholecalciferol at 1 or 100 nM significantly inhibited parathyroid hormone secretion when medium calcium concentration was normal (1.5 mM), high (3.0 mM), and low (1.0 mM). Maximum inhibition ranged from 19 to 74%; inhibition was generally seen after 2 h of incubation; and inhibition was sustained or progressive thereafter. Vitamin A, 0.1 muM, caused a marked stimulation of parathyroid hormone secretion. 1,25-Dihydroxycholecalciferol at 1 nM markedly reduced (44%) the effect of vitamin A to stimulate parathyroid hormone secretion. This effect of 1,25-dihydroxycholecalciferol was maximal at 1 h and persisted thereafter. Another steroid, hydrocortisone, 10 muM, did not inhibit parathyroid hormone secretion, suggesting that the 1,25-dihydroxycholecalciferol effect was not a nonspecific inhibitory effect on parathyroid cells. Because other workers have shown that parathyroid hormone directly stimulates 1,25-dihydroxycholecalciferol secretion, our results are consistent with the concept that there is a feedback loop where parathyroid hormone directly stimulates secretion of 1,25-dihydroxycholecalciferol, which in turn directly inhibits secretion of parathyroid hormone.     

高磷对大鼠甲状旁腺组织及原代培养细胞的刺激作用

目的观察高磷刺激下,离体的大鼠甲状旁腺（PTG）组织和培养的原代甲状旁腺细胞的PTH分泌变化规律。方法解剖显微镜下切取28只Wistar雌性大鼠的两侧甲状旁腺,分别行组织和原代细胞培养。应用组织学形态学、PTH检测对甲状旁腺组织及原代培养细胞进行鉴定,并观察PTH的分泌规律。甲状旁腺组织及原代培养细胞组各分为正常磷、正常钙组（TN,CN,Pi1.0mml/l,Ca 1.25mmol/L）,高磷、正常钙组（TH,CH,Pi3.5mmol/L,Ca 1.25mmol/L）。隔天换液,分别于培养0h、4h、12h、24h、48h、72h、5d、7d、9d留取培养上清液,检测iPTH。结果本实验切取的大鼠甲状旁腺组织和培养的甲状旁腺细胞经组织形态学及iPTH检测证实。在正常磷、正常钙的培养液中,甲状旁腺组织和培养的原代甲状旁腺细胞PTH分泌曲线相似,均在48h达到分泌高峰。在高磷、正常钙的培养液中甲状旁腺细胞PTH分泌与正常磷、正常钙组同一时间比较,差异无统计学意义（P〉0.05）。培养的甲状旁腺组织在高磷刺激下,PTH水平明显升高,最高达基础值的10倍以上（48h）,除H0外,高磷、正常钙组（TH组）PTH与正常磷、正常钙组（TN组）同一时间比较差异均具有统计学意义（P〈0.001）。结论体外研究显示：高磷可以刺激甲状旁腺组织PTH分泌增加,而对分散的细胞无作用。进行高磷对甲状旁腺功能调节的体外研究,以选择甲状旁腺组织为宜,且时间选择在48h内。     

Adrenergic interaction in mucin secretion of rat submandibular gland in vitro

Previous studies have reported that maximally effective concentrations of the "mixed" alpha and beta adrenoceptor agonists, epinephrine and norepinephrine, cause greater amounts of mucin secretion than the "pure" beta adrenoceptor agonist, isoproterenol, and that this response requires extracellular calcium. The purpose of the present study was to examine directly the nature of the effect of the putative pure alpha adrenoceptor agonist, phenylephrine, on isoproterenol-induced mucin secretion and the role of extracellular calcium in this interaction. We used a graphical method which provides a procedure for determining whether interaction between two drugs is additive, antagonistic or synergistic to analyze this interaction. Submandibular glands were removed from male rats, divided into sections and placed in modified Hank's balanced salt solution. Mucin secretion was measured as acid precipitable disintegrations per minute after labeling the submandibular gland with [14C]glucosamine. Phenylephrine caused a small but significant secretion of mucin which was blocked partially by phentolamine and completely by propranolol. Phenylephrine caused a significant shift of the isoproterenol concentration-response curve to the left of the theoretical curve expected if two drugs act additively. Mucin secretion induced by isoproterenol alone was independent of extracellular calcium concentration; however, the combination of isoproterenol and phenylephrine caused a concentration-dependency on extracellular calcium.     

Epidermal growth factor stimulates synthesis and secretion of mucus glycoproteins in human gastric mucosa

1. The effects of epidermal growth factor on the synthesis and secretion by human gastric mucosa of radiolabelled mucus glycoprotein have been studied in organ culture. 2. Addition of epidermal growth factor at a concentration of 5 or 10 ng/ml increased the synthesis of glycoprotein from 84 +/- 16.2 d.p.m./micrograms of protein to 132 +/- 19.5 and 156 +/- 20.2 d.p.m./micrograms of protein, respectively. The same doses increased secretion from 73.75 +/- 17 d.p.m./micrograms of protein to 114.0 +/- 26.4 and 141.5 +/- 31.4 d.p.m./micrograms of protein (P less than 0.001). 3. As glycoproteins are the main constituent of the mucus defence barrier, epidermal growth factor-stimulated mucus production may contribute to gastric cytoprotection.     

Potent mitogenic effects of parathyroid hormone (PTH) on embryonic chick and rabbit chondrocytes. Differential effects of age on growth, proteoglycan, and cyclic AMP responses of chondrocytes to PTH.

The effect of PTH on chondrocyte proliferation as a function of cartilage age was examined. PTH[1-34] induced a 12- to 15-fold increase in the efficiency of colony formation in soft agar by chondrocytes from embryonic 13- to 19-d-old chickens and fetal 25-d-old rabbits with a 10-fold increase in their DNA content. It also caused a 2.5-fold increase in [3H]thymidine incorporation into DNA in fetal 25-d-old rabbit chondrocytes. No mitogenic responses to PTH were observed, however, in postnatal 7- to 21-d-old chick chondrocytes or postnatal 21-d-old rabbit chondrocytes. This age dependency was observed only with PTH: fibroblast growth factor, epidermal growth factor, and insulin stimulated chondrocyte proliferation irrespective of cartilage age. The absence of a mitogenic effect in postnatal chondrocytes was not due to a decrease in number or a reduction in affinity of receptors for PTH. PTH also increased [35S]sulfate incorporation into proteoglycans and the cyclic AMP level in fetal and postnatal chondrocytes, but at 100-fold higher concentrations (10(-8)-10(-7) M) than those (10(-10)-10(-9) M) required for the stimulation of cell division. These results suggest that PTH is a potent mitogen for embryonic chondrocytes, and that its mitogenic effect disappears selectively after birth.     

Direct release of parathyroid hormone fragments from functioning bovine parathyroid glands in vitro.

To determine the origin of circulating parathyroid hormone fragments, hormonal peptides released from bovine parathyroid tissue in a physiologically responsive in vitro "perifusion" system were analyzed by gel exclusion chromatography and region-specific radioimmunoassays. When exposed to low Ca++, the tissue released large quantities of intact hormone (parathyroid hormone 1--84) as well as amino- and carboxyl-terminal fragments. Fragments of the hormone were also released when the tissue was exposed to high Ca++, but the carboxyl fragments comprised a much greater proportion of the hormonal peptides released. Control experiments indicated that fragmentation of the hormone occurred within the gland and not after it was secreted. These experiments provide direct evidence, therefore, that release of fragments from the parathyroid gland may contribute to the immunologic heterogeneity of the hormone in the circulation.     

Tumor promoter phorbol myristic acetate stimulates immunoglobulin secretion correlated with growth cessation in human B lymphocyte cell lines.

Immunoglobulin production by lymphoblast cell lines was studies using protein A-red blood cell plaque formation to detect individual secreting cells. Immunoglobulin (Ig) secretion by 6 of 12 human B-cell lines tested could be stimulated up to twentyfold by phorbol myristic acetate (PMA) at subtoxic concentrations of 10-1000 ng/ml depending on the line. Stimulation was found with both IgM and IgG cell lines. No switch of Ig class synthesis was found in the cell lines as a result of PMA incubation. Increase in Ig secretion was closely associated with cessation of growth resembling induction of terminal differentiation in the cells. PMA induction of Ig secretion in B lymphocytes from normal peripheral blood requires the cooperation of T cells. PMA stimulation of certain cell lines reported here suggests that the lines are late in the differentiation pathway to plasmacyte and can be easily triggered to secrete Ig by membrane-altering agents.     

Synthetic human parathyroid hormone-like protein stimulates bone resorption and causes hypercalcemia in rats.

Parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs) have been implicated as one of the calcemic, bone-resorbing agents in patients with humoral hypercalcemia of malignancy. We report the synthesis of an amino-terminal hACSP fragment, Tyr36 hACSP (1-36) amide. The synthetic hACSP is a potent agonist of renal membrane adenylate cyclase (Km, 1.7 X 10(-10)) and of bone cell adenylate cyclase (Km 1 X 10(-9)M). It is a potent bone-resorbing agent in vitro, stimulating 45Ca release from fetal rat long bones at a concentration of 10(-9) M. When infused via osmotic minipumps into rats, it is also a potent calcemic factor in vivo, inducing a rise in serum calcium from (mean +/- SD) 10.6 +/- 0.6 to 19.7 +/- 3.2 mg/dl when infused at 1.4 micrograms/h and from 9.9 +/- 0.7 to 11.4 +/- 1.2 mg/dl when infused at 0.14 micrograms/h. These findings indicate that biologically active hACSP fragments can be synthesized. One such synthetic peptide possesses the in vitro and in vivo bioactivities demonstrated in native, tumor-derived hACSPs. It is also a potent calcemic, bone-resorbing agent.     

Z-velocity in screening for intrauterine growth restriction.

OBJECTIVES: Ultrasound scans provide the basis for detection of intrauterine growth restriction (IUGR) but often fail to distinguish IUGR from small-for-gestational age (SGA) fetuses. This study introduces the concept of Z-velocity, calculated as changes in Z-scores over time, as an additional criterion in the diagnosis of IUGR. METHODS: A computer program simulated 50 000 fetal abdominal circumference (FAC) scans based on published growth formulae. False-positive rates were calculated to determine optimal scan time and scan intervals. Using an independent simulation of 32 500 FAC scans, the two methods were compared using receiver-operating characteristics (ROC) curve analysis. RESULTS: ROC showed areas under the curve of > 0.74 over the complete range of scan intervals. The positive predictive value of growth arrest as the only diagnostic criterion was, however, too low to recommend it as an exclusive or the first diagnostic criterion. CONCLUSIONS: Z-velocity can be used to decide whether further investigations for growth abnormality are required in fetuses that fall below the 10(th) percentile. The gain of combined diagnostic approaches should be calculated from large databases that include the neonatal ponderal index as the gold standard.     

Beta endorphin selectively stimulates aldosterone secretion in hypophysectomized, nephrectomized dogs.

We examined the effects of synthetic human beta-endorphin (beta END) and a stable methionine (Met)-enkephalin analogue on aldosterone and cortisol secretion rates in anesthetized, hypophysectomized, and nephrectomized dogs and compared them to those of (1-39) ACTH. The circulation of the adrenal glands was completely isolated on the arterial and venous sides (Hilton Pouch). The peptides were infused to deliver 3 pmol/min into the aortic "pouch." Blood was collected from the vena caval pouch, which received blood only from the adrenal gland. Secretion rates of aldosterone and cortisol were calculated as the product of adrenal blood flow and venous steroid concentration. Duplicate steroid measurements were obtained during a control period, at 10, 30, and 50 min of peptide infusion and during a postcontrol period. BetaEND increased aldosterone secretion rate from 2.4 +/- 0.5 ng/min (mean +/- SEM) to 3.2 +/- 0.9 ng/ min at 10 min (N.S.), 8.2 +/- 2.5 ng/min (P less than 0.05) at 30 min and 11.0 +/- 3.7 ng/ min (P less than 0.05) at 50 min of infusion. Cortisol secretion rate was not affected by infusion of betaEND. Infusion of the stable Met-enkephalin analogue D-alanine2; Metphenylalanine4, Met(O)-enkephalin-ol or saline alone had no effect on aldosterone or cortisol secretion rates. ACTH infusion increased mean aldosterone secretion rate by approximately 215% and significantly stimulated cortisol secretion rate. These results indicate that beta END selectively stimulates aldosterone secretion with a potency similar to that of an equimolar dose of ACTH.     

Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with high affinity thus stabilizing the F-actin cytoskeleton by preventing depolymerization, an event necessary for dynamic reordering of actin microfilaments. NBD-phallicidin loading is not cytotoxic as assessed by lactic dehydrogenase release, protein synthesis, transepithelial resistance, and the ability of the loaded cells to pump Na+ in an absorptive direction in response to the apical addition of a Na+ ionophore. However, cAMP-elicited redistribution of F-actin and the cAMP-elicited Cl- secretory response are both markedly impaired in NBD-phallicidin preloaded T84 cells. In contrast, the carbachol-elicited Cl- secretory response (Ca++ mediated) is not attenuated by NBD-phallicidin preloading nor is it accompanied by redistribution of F-actin. These findings suggest that the cAMP-elicited cytoskeletal redistribution we describe is an integral part of cAMP-elicited Cl- secretion in T84 cells.     

Triiodothyronine stimulates maturation of porcine growth-plate cartilage in vitro.

We studied the effect of triiodothyronine (T3) on mammalian growth-plate cartilage in vitro. Growth-plate cartilages from fetal pigs scapulae were incubated for 3 to 7 d in serum-free medium alone or medium containing T3. Alkaline phosphatase activity, a marker of hypertrophied chondrocytes, was increased in T3 (10 nM)-treated growth-plate cartilage 152 +/- 36% above that of cartilage incubated in medium alone after 3 d of incubation, and 324 +/- 47% after 7 d of incubation. There was a dose-response increase in alkaline phosphatase activity to T3 over the range of 0.01-10 nM. The rise in alkaline phosphatase activity was specific for T3 since growth-plate cartilage alkaline phosphatase activity was not increased by cortisol, insulin, parathyroid hormone, or 5% fetal calf serum. Histological studies of growth-plate cartilage showed that T3 in a concentration-dependent manner increased the width of the zone of maturation (hypertrophied chondrocytes). Histochemical staining for alkaline phosphatase activity demonstrated that T3 caused the recruitment of new cells into the zone of maturation. T3 also stimulated incorporation of L-[3H]leucine into protein and 35SO4 into proteoglycan in growth-plate cartilage. In contrast, T3 did not increase alkaline phosphatase activity or radiolabeled precursor incorporation into nongrowth-plate scapular cartilage. These studies demonstrate that T3 directly stimulates maturation and, to a lesser degree, growth-related processes in fetal mammalian growth-plate cartilage.     

C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha

We have demonstrated that purified C5a is a potent stimulus to human PBMC secretion of TNF-alpha, IL-1 beta, and IL-1 alpha, which proceeds in a dose-dependent fashion. At a given concentration of C5a, TNF-alpha and IL-1 beta secretion did not differ significantly; both were secreted in significantly greater quantity than IL-1 alpha. Clinical conditions such as Gram-positive and Gram-negative bacterial infections, trauma, and immune complex diseases activate complement. Through the mediation of TNF and IL-1 secreted in response to C5a, these diverse disorders can share common features of fever, coagulopathy, acute phase protein production, and disordered metabolism.     

Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo.

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two- to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N-(3-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and gamma chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro two- to fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cell lines.