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Background/aims: The characteristics in patients with hepatocellular carcinoma who survive more than 10 years after hepatectomy remain unclear., Methodology: Eighty-five cases of hepatocellular carcinoma were retrospectively identified as short-term survivors (S-group: <5 years, n=41), medium-term survivors (M-group: > or =5 and <10 years, n=25), and long-term survivors (L-group: > or =10 years, n=19) to examine the clinicopathologic factors for the 10-year survival after curative hepatectomy., Results: In the L-group, platelet count and albumin level were higher and total bilirubin level was lower than those in the S-group. In the S-group, the total bilirubin level was higher and vascular invasion was more frequent than those in the M-group. Multiple regression analysis revealed that only host-related factors such as age, albumin level, and total bilirubin level were selected as significant factors to determine the 10-year survival. However, no tumor-related factor was selected., Conclusions: The present study suggests that the important factor linked to the 10-year survival of hepatocellular carcinoma patients is the host-related factor, but not the tumor-related factor. Especially, younger age, higher albumin level, and lower total bilirubin level are quite important to determine the 10-year survival.

Background/aims: Laparoscopic splenectomy is now increasingly being performed. However, controversy remains regarding the effectiveness of a laparoscopic splenectomy for patients with portal hypertension., Methodology: Seventy-three patients with portal hypertension who underwent a laparoscopic splenectomy from February 1992 until October 2000 were reviewed and the effectiveness of the procedures for portal hypertension was evaluated. Forty-two patients had esophagogastric varices and twenty had a concomitant hepatocellular carcinoma. The indications for surgery were bleeding tendency due to thrombocytopenia (n = 40), difficulty in receiving treatment for hepatocellular carcinoma due to thrombocytopenia (n = 18), and sclerotherapy-resistant esophagogastric varices (n = 15)., Results: A laparoscopic splenectomy was successfully performed in all the patients. The rate of conversion to conventional open surgery was 9.6% (7/73). The mean operative time was 210.1 +/- 101.9 minutes, and the estimated blood loss was 374.7 +/- 352.4 mL. There were no cases of mortality, and morbidity was encountered in 11.0% of patients. The increase in the platelet count correlated significantly to the spleen weight (P < 0.001). The platelet count had been maintained at over 10 x 10(4)/mm3 for over three years. Eighteen patients with hepatocellular carcinoma successfully underwent treatment for hepatocellular carcinoma after surgery and no recurrence of esophagogastric varices was encountered., Conclusions: A laparoscopic splenectomy resulted in the successful secondary treatment of hepatocellular carcinoma and esophagogastric varices. Portal hypertension was not a contraindication. A laparoscopic approach is therefore the procedure of choice for a splenectomy in portal hypertension.

Background/aims: This study investigates the effect of prostacyclin, which is thought to be involved in the hemodynamic circulation, on the gastric mucosa of rats with portal hypertension., Methodology: Various gastric functions were evaluated in portal vein ligated rats after the intraperitoneal administration of either a placebo or prostacyclin for 7 days., Results: The gastric mucosal damage induced by the instillation of 90% ethanol was significantly greater in the prostacyclin-treated group than in placebo-treated group. The portal pressure was similar in both groups. There was no significant difference between the two groups in plasma concentration of 6-keto-PGF1a (a stable metabolite of prostacyclin), whereas the mucosal content of 6-keto-PGF1a was significantly higher in the prostacyclin-treated group than in the placebo-treated group. Prostacyclin pretreatment significantly increased the gastric mucosal blood flow, estimated by laser-Doppler flowmetry, and the hemoglobin content of the gastric mucosa, measured by reflectance spectrophotometry, whereas the oxygen content remained unchanged., Conclusions: We speculate that the increased gastric mucosal perfusion induced by a high content of prostacyclin in the portal hypertensive gastric mucosa may play a role in the pathogenesis of ethanol-induced gastric mucosal damage.

We describe the case of a 51-year-old woman with primary biliary cirrhosis who developed hyperbilirubinemia with transient liver dysfunction after undergoing endoscopic variceral ligation to control hemorrhaging from esophageal varices. After undergoing a variceal ligation, the serum total bilirubin increased from 4.0 mg/dL to 9.5 mg/dL, and the degree of liver failure worsened. She finally had to undergo a liver transplant. We discuss the mechanism of hyperbilirubinemia after endoscopic variceal ligation.

Background/aims: Gastroduodenal ulcer is a very common illness in Japan. As the number of elderly persons in Japan increases the same as in Europe and America, the number of such patients requiring a gastroduodenal emergency operation has also increased. Regarding the complications of peptic ulcer, a perforation remains the most important fatal complication. The aim of this study is to investigate the operative risk factors and the long-term recurrence rates and to define the optimal surgical procedures in emergency situations in elderly patients., Methodology: From April 1988 through March 1997, 130 patients over 70 years of age with a perforated gastroduodenal ulcer (a duodenal ulcer perforation in 50 patients and a gastric ulcer perforation in 80 patients) were operated on in an emergency situation in our clinic. We investigated the following items; medical illness, preoperative risk factor, optimal surgical procedure, postoperative organ failure and the cumulative recurrence-free rates after surgical treatment., Results: A significant correlation with mortality was observed in patients with established comorbidity in the following organs: lung (P = 0.03), heart (P = 0.02), kidney (P = 0.04), and diabetes (P = 0.03). The highest postoperative mortality rate was recorded in patients who underwent a simple closure of a duodenal ulcer perforation (4 patients; 26.7%), while the lowest postoperative mortality rate was recorded in patients who underwent a simple closure and vagotomy of a duodenal ulcer perforation (3 patients; 12.5%). In gastric ulcers, the mortality rate in patients with a gastrectomy was significantly higher than in patients with a simple closure. The practical application of the three risk factors (preoperative shock, delay to surgery over 24 hours, and medical illness) was shown by the progressive rise in the mortality rate with the increasing number of risk factors. Based on the 5 postoperative years after treating a perforated duodenal ulcer, the cumulative recurrence rate after a simple closure (63.6%) was significantly higher than that after a simple closure and vagotomy (38.1%) (n = 0.02) or after gastrectomy (0%) (P < 0.001). At 5 years postoperatively, the cumulative recurrence rate after a simple closure (41.2%) was significantly higher than that after a gastrectomy (15.9%) (P < 0.01)., Conclusions: In conclusion, in an emergency situation, elderly patients are in a highly unfavorable prognostic condition due to their advanced age, and comorbidity, which thus leads to poorer results, not only worldwide, but also in Japan. Based on our findings, in duodenal ulcer cases, a simple closure and vagotomy is recommended because of its low mortality and minimal stress, except for cases with a giant perforation measuring over 20 mm in diameter at the perforation hole or with severe duodenal stenosis. In stomach ulcer cases, a gastrectomy may be recommended because of its low recurrence rate.

Two cases undergoing a laparoscopic splenectomy for the treatment of a splenic artery aneurysm are herein reported. This lesion is relatively rare. Surgical treatment is indicated for such cases since approximately 10% of these aneurysms tend to rupture which thus results in fatal hemorrhaging. Both cases demonstrated aneurysms measuring more than 2 cm in diameter based on the ultrasonography, computed tomography and celiac angiography findings and, as a result, a laparoscopic splenectomy was thus prophylactically performed. This procedure is the preferred technique for high risk patients, such as those with chronic renal failure, as observed in case 1, since patients can be spared the disadvantages of undergoing a laparotomy.

Background/aims: Recent advances in technical instruments have resulted in increased safety and simplicity in laparoscopic surgery. The purpose of this article is to introduce our latest operative techniques for laparoscopic splenectomy., Methodology: The patient is placed in the right semidecubitus position and the gastrosplenic ligament including the short gastric vessels was performed by using an ultrasonically activated scalpel. The splenic artery and vein were resected at the splenic hilum with an autosuture device. The electromechanical morcellator was used to remove the spleen., Results: The laparoscopic splenectomy was successfully performed in all 74 patients from 1992-1997. There was no deaths related to the operation. Conversion to open surgery with a small incision of 5 cm was required in one patient with advanced liver cirrhosis and portal hypertension and 45 patients with portal hypertension., Conclusions: A laparoscopic splenectomy is considered to be a safe and feasable modality for the treatment for hematologic disorders of both the spleen and other benign tumors.

Background/aims: The present study was an attempt to clarify whether only serum albumin inactivates ethanolamine oleate (EO) in vivo and whether EO has an adverse effect on the lung, kidney, and liver in the presence of analbuminemia., Methodology: Fifty-five female Nagase Analbuminemia Rats (NAR) and 55 female Sprague-Dawley Rats (SDR) were injected with 5% EO in the left femoral vein. Changes in respiratory, renal, and hepatic function and the extent of hemolysis following the injection of the EO were examined., Results: In both groups, a transient but significant increase was seen in serum hemoglobin at up to 1 hour after injection of EO. No significant changes in PaO2, PaCO2, blood urea nitrogen, or serum creatinine following injection of EO were seen in either group. The total bilirubin significantly increased within 30 min in the SDR group, but this increase was prolonged for 5 days in the NAR group. Glutamic pyruvic transaminase (GPT) increased for 12 hours in the NAR group, while no change was seen in the SDR group., Conclusions: These observations suggest that EO may have hepatotoxic effects, that serum albumin may afford protection, and that other humoral substances which inactivate EO, including serum globulin, may be present.

Background/aims: One of the major hurdles to overcome in the development of laparoscopic surgery has been finding effective extraction techniques for the removal of large tissue masses. The electromechanical morcellator makes it easy to remove relatively large sections of tissue from the abdomen through the existing incisions. To determine the safety and efficacy of the new morcellator in laparoscopic splenectomy, our preliminary experiences using the morcellator were reviewed and retrospectively compared with the results in patients who previously underwent conventional extraction techniques., Methodology: From February 1992 to March 1996, 31 patients underwent laparoscopic splenectomy. In the last eight patients, the new morcellator was used to remove the spleen, while in the remaining 23 patients, a laparoscopic splenectomy was performed using the conventional extraction techniques., Results: In the last eight patients who underwent laparoscopic splenectomy, the newly devised morcellator was successfully used without any complications. The spleen could be removed from the abdominal cavity in an average of 15.9 +/- 10.4 minutes using the morcellator, while the average was 45.7 +/- 15.4 minutes using conventional techniques. It was not necessary to extend the skin incision in patients with the morcellator, while, on the other hand, a 2-cm extension of the existing skin incision was always required to remove the spleen in the latter method., Conclusion: The morcellator was found to be an effective device which can safely, efficiently and rapidly remove tissue masses. This procedure is therefore considered to be one feasible way to solve the age-old problem of intracorporeal morcellation.

This report describes a 38-year-old man with massive gastrointestinal bleeding from jejunal varices. He had been previously diagnosed to have idiopathic portal hypertension and esophageal varices, and had undergone an esophageal transection 8 years earlier. The pre-operative diagnosis was a suspected hemorrhage from the small intestine as visualized by 99mTc-HSAD scintigraphy (technetium 99m-labeled human serum albumin D-type) and was not considered to be repeated massive lower GI tract bleeding. An exploratory laparotomy was performed, and intra-operative endoscopy revealed active bleeding from the jejunal varices. A partial resection of the small intestine resulted in a complete resolution of the bleeding. A review of the literature thereafter disclosed twelve previously reported cases of jejunal variceal bleeding.

Background/aims: The long-term results of a combination therapy of sclerotherapy with percutaneous transhepatic obliteration (PTO) was reviewed in 37 patients with large gastric varices. The study suggests that sclerotherapy combined with PTO can safely and effectively eradicate large gastric varices as an alternative approach to surgery. Complete eradication has also been shown to prevent the recurrence of gastric varices., Material and Methods: The long-term results of a combination therapy of sclerotherapy with PTO was reviewed in 37 patients with large gastric varices., Results: Complete eradication of the gastric varices was obtained in 26 patients (70.3%). There was no recurrence of the varices in whom complete eradication had been obtained, but in 11 patients with incomplete eradication gastric varices recurred during the mean follow-up period of 37.5 months. Bleeding occurred in 4 after the treatment and one patient died of massive bleeding from the recurrent gastric varices. There were no major complications with the procedure except for one who had a peripheral pulmonary arterial embolization as a result of a steel coil used for the PTO., Conclusion: This study suggests that sclerotherapy combined with PTO can effectively and safely eradicate large gastric varices and is an alternative approach to surgery for treating patients with gastric varices. It is also shown that complete eradication leads to a prevention of recurrence of gastric varices.

To study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder, which is caused by deficiency or decrease of the platelet GPIb/IX/V complex. Analysis of two patients with BSS by flow cytometry of the blood revealed different expression patterns of the components of the GPIb/IX/V complex. In case 1, GPIX was completely absent but residual amounts of GPIb alpha and GPV were detectable; in case 2, GPIb alpha was completely absent. We amplified the coding regions of GPIb alpha, GPIb beta, GPV, and GPIX from the patients' genomic DNA with the polymerase chain reaction (PCR) and sequenced the PCR products. in case 1, we identified a point mutation in the GPIX coding region that changes the codon for tryptophan-126 (TGG) to a nonsense codon (TGA). In case 2, we found a deletion of nucleotide within seven adenine repeats at the position of 1932 to 1938 in the coding region of GPIb alpha, which causes a frame shift that results in 58 altered amino acids and a premature stop codon. These genetic changes alter the transmembrane domain of GPIX or GPIb alpha and, therefore, would prevent proper insertion of the proteins in the plasma membrane. Thus, abnormality of a single component protein (GPIX or GPIb alpha) alters the assembly of the GPIb/IX/V complex and causes heterogeneous surface expression of GPIb alpha, GPV and GPIX.

A 56-year-old Japanese man with liver cirrhosis was admitted to Kyushu University Hospital in September, 1986 for the treatment of large esophageal varices. Endoscopy revealed four tortuous folds of large esophageal varices, extending proximally from the esophagocardial junction to 34 cm from the dental arch. Endoscopic injection sclerotherapy was performed on the lower esophagus using 5% ethanolamine oleate, and esophageal varices were completely eradicated in 5 sessions with a total of 70 ml of sclerosant. In March 1991, 4 years and 6 months after the treatment, endoscopy revealed a mild redness and an irregular surface 33 cm from the dental arch. The histologic diagnosis was squamous cell carcinoma. The possible relationship between sclerotherapy and the development of esophageal carcinoma should be considered.

Background/aims: The most common cause of failure of sclerotherapy is recurrent bleeding before eradication is complete. We investigated factors which would make feasible prediction of cases where esophageal varices would be more difficult to eradicate., Patients and Methods: Seven hundred and seventy patients underwent endoscopic injection sclerotherapy at Kyushu University Hospital from January, 1982 to June, 1989. For 580 of these patients we used the same sclerosant and a transparent overtube. For 19 of 580 patients over two months were needed to eradicate the varices (group 2), while eradication was complete in less than one month in 64 patients (group 1)., Results: There was a tendency toward a lower platelet count and a higher indocyanine green retention rate in group 2, but with no statistically significant difference. The number of sessions required for eradication of the varices was 8.1 +/- 2.5 and the total volume of sclerosant used was 98.2 +/- 62.3 ml in group 2, and 3.0 and 47.0 +/- 10.9 ml, respectively, in group 1 (p < 0.01). There was no significant difference in the number of sessions between the patients with large-sized and moderate-sized varices. Based on the extent of cephalad collateral vessels on the venous phase of celiac or superior mesenteric angiography, the vascular pattern could be classified into three types; Grade III, the most developed type was present in 100% and 57.1% on celiac and superior mesenteric angiography in group 2, while the rates were 11.1% and 5.6% in group 1 (p < 0.05)., Conclusions: This retrospective study shows that in patients with enormously enlarged cephalad collateral vessels it may be difficult to eradicate the varices, and in such cases, preoperative portography is most useful to predict whether or not esophageal varices can be eradicated.

In our ongoing studies on the hemodynamics of splenic artery aneurysms (SAA) in portal hypertension, 32 patients with portal hypertension were examined by duplex sonography and angiography. Four out of 32 (12.5%) had SAA, and SAA(+) and SAA(-) groups were formed. On duplex sonography, splenic venous blood flow in the SAA(+) group (1058 +/- 211 ml/min) increased more than in the SAA(-) group (423 +/- 173 ml/min, p < 0.01), but there was no significant difference in portal venous flow. On angiography, there were significant differences in the diameters of the splenic arteries between the SAA(+) group (7.5 +/- 0.5 mm) and the SAA(-) group (5.6 +/- 1.0 mm, p < 0.01). All patients (100%) in the SAA(+) group had prominent portosystemic shunts, while 8 out of 28 (28.6%) in the SAA(-) group had these shunts (p < 0.05). Thus, there seems to be a splenic hyperkinetic state in patients with SAA and portal hypertension, a finding which may closely relate to the pathogenesis of SAA.

In attempts to avoid hemolytic nephropathy following injection of 5% ethanolamine oleate during endoscopic injection sclerotherapy for esophageal varices, the relationship between serum levels of albumin and hemolysis induced by ethanolamine oleate was investigated. The rate of hemolysis at various concentrations of ethanolamine oleate and albumin was examined using a spectrophotometer in ten cirrhotic patients and five healthy volunteers (controls). Hemolysis induced by ethanol-amine oleate increased in an ethanolamine oleate concentration-dependent fashion (p < 0.01) but was dose-dependently inhibited by albumin (p < 0.05). The state of liver function was unrelated to the rate of hemolysis. The relationship between hemoglobinuria and serum albumin levels in seventy-nine cirrhotic patients with esophageal varices treated by endoscopic injection sclerotherapy with ethanolamine oleate was also examined. Hemoglobinuria was evident in 20 out of 24 patients (83.8%) in whom the serum albumin level was less than 3.0 g/dl, and in 24 out of 55 (43.6%) in whom it exceeded 3.0 g/dl, the difference being statistically significant (p < 0.01). On the day after endoscopic injection sclerotherapy, creatinine clearance fell from 81.6 +/- 33.8 m/l min to 60.1 +/- 31.4 ml/min in the hemoglobinuria-positive patients (p > 0.01) and two went into acute renal failure. The creatinine clearance ranged from 79.5 +/- 27.9 ml/min to 83.0 +/- 39.9 ml/min in the hemoglobinuria-negative patients (p < 0.1). In the light of this evidence we correct the serum albumin level cut off point to 3.0 g/dl prior to endoscopic injection sclerotherapy in order to maintain renal function.

Sixty-six patients with portal hypertension and esophageal varices due to liver cirrhosis were randomized to receive either 5% ethanolamine oleate (EO) or 5% EO plus 1% polidocanol (EOP) as a sclerosant for endoscopic injection sclerotherapy (EIS). The two groups were well matched with regard to age, sex and the severity of liver disease. In no patient in the two groups was there any major complication, such as esophageal perforation or esophageal bleeding. Eradication of esophageal varices was attained with an average of 4.7 and 4.3 sessions of endoscopic injection sclerotherapy in the ethanolamine oleate and polidocanol groups, respectively. Data on one patient in the ethanolamine oleate group had to be excluded because he left the hospital after 2 sessions of endoscopic injection sclerotherapy. Esophageal ulcers occurred earlier in the polidocanol group (after an average of 2.8 weeks) than in the ethanolamine oleate group (3.8 weeks), the difference being statistically significant (P < 0.01). The rate of occurrence of esophageal stricture requiring more than 2 sessions of bougienage was significantly (P < 0.01) higher in the polidocanol group (16/33, 48%) than in the ethanolamine oleate group (4/32, 12%). This study suggests that the two sclerosants have equal efficacy for treating patients with esophageal varices. With polidocanol there was ulceration and stricture in the distal esophagus.

Forty-eight out of six hundred and thirty patients with portal hypertension undergoing a celiac angiography series were diagnosed as cases of splenic artery aneurysm during the period 1977-1988. The case-control study of patients with portal hypertension with splenic artery aneurysms and those without was designed to characterize the angiological features. The splenic arterial flow was assessed by measuring the radii of the splenic arteries on celiac arteriograms. In the portal hypertensive patients with splenic artery aneurysms, the splenic artery was larger (p < 0.05) and the splenic arterial flow greater (p < 0.05), and these patients were in a more hyperkinetic state, than were those with no splenic artery aneurysm. The study suggests that splenic artery aneurysms in cases of portal hypertension may be the consequence of a hyperkinetic state in the spleen.

Platelet membrane glycoprotein Ib (GPIb) functions as receptors for thrombin and von Willebrand factor (vWF) in the presence of ristocetin. To precisely locate the domains on GPIb interacting with vWF and thrombin, we prepared several peptides that have amino acid sequences analogous to that of the GPIb alpha-chain and examined their effects on ristocetin-induced (vWF-dependent) and thrombin-induced platelet aggregations. A peptide extending from residues Asp235 to Lys262 showed the strongest inhibitory effect on ristocetin-induced platelet agglutination, and a group of overlapping peptides composed of 24-28 amino acid residues representing sequences extending from Phe216 to Asp274 was found to inhibit platelet aggregation induced by thrombin. Other peptides did not inhibit platelet aggregations. Moreover, the binding to platelets of the monoclonal anti-GPIb antibody (TM60) which had been shown to inhibit both ristocetin- and thrombin-induced platelet aggregations was strongly inhibited by a peptide extending from Asp249 to Asp274. These data demonstrate that the vWF-binding domain exists in a small region between residues Asp235 and Lys262; the thrombin-interacting domain, in contrast, is located between residues Phe216 and Ala274, with a possible center of interaction in the sequence from Phe216 to Thr240 on the GPIb alpha-chain, and thrombin binding requires a relatively strict conformation in this domain.

Pretreatment of platelets with chymotrypsin dose-dependently decreased glycoprotein (GP)-Ib amounts as measured by SDS-PAGE, ristocetin-induced agglutination and platelet electrophoretic mobility (EPM). Decrease in platelet EPM in response to 0.75 mg/ml ristocetin alone were 7.0 +/- 2.3 and 6.8 +/- 4.3% (M +/- S.E., n = 6) for control and chymotrypsin-treated platelets, respectively (p greater than 0.2). Von Willebrand factor (vWF) alone had no effect on platelet EPM. However, in the presence of 0.75 mg/ml ristocetin, added vWF (2.9 micrograms/ml) caused a further 6.3 +/- 3.8% decrease in control platelet EPM, but caused no significant decrease in the enzyme-treated platelets (p less than 0.05). In the presence of 0.3 mg/ml ristocetin, added vWF (2.9-14.5 micrograms/ml) caused a small but significant decrease in control platelet EPM, but caused no significant decrease in the enzyme-treated platelets. These findings suggested that the GP-Ib carrying negative charge decreased by binding of vWF might facilitate a mutual approach of the GP-Ib molecules and bridge formation by vWF between different platelets.

To determine a thrombin-binding site on GPIb alpha on platelet membrane, we have examined the binding activities of tryptic or chymotryptic fragments of purified GPIb alpha to a monoclonal antibody against GPIb (TM60) and thrombin using (immuno)affinity chromatography. When purified GPIb alpha was digested with trypsin, two fragments (94-kDa, and 43-kDa) were obtained. The 43-kDa fragment was shown to bind to both affinity columns of TM60- and thrombin-Affi-Gel, while the 94-kDa fragment did not bind to either Affi-Gel columns. When trypsin fragments were incubated with TM60 and then applied to the column of thrombin-Affi-Gel, neither fragments were bound to the column. When the same experiment was performed using chymotrypsin, three fragments (94-kDa, 45-kDa and 39-kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45-kDa and 39-kDa) were bound to the column. After incubation of these fragments with TM60, neither bound to the thrombin column. These results indicate (i) that the epitope for TM60 is located near, or on the thrombin-binding site of GPIb alpha, and (ii) that the thrombin-binding site is located on the tail portion of GPIb alpha, especially on a chymotrypsin cleavage site.

The relationship between platelet surface negative charge and hyperfunction was examined by determining electrophoretic mobility (EPM), aggregability, and sialic acid of platelets in prostatic cancer, prostatic cancer with estrogen, prostatic cancer with estrogen and aspirin, prostatic hypertrophy, and healthy aged males. Estrogen treated prostatic cancer patients had significantly higher platelet EPM. A good linear correlation was found between sialic acid and EPM (r = 0.97, p less than 0.001). EPM was negatively correlated with primary aggregations by adrenaline and ADP but not with secondary or maximum aggregations, suggesting increased surface negative charge may inhibit primary aggregation. Estrogen and platelet population changes influenced surface negative charge. Neuraminidase removal of platelet surface sialic acid resulted in dose-dependent decreases of EPM which paralleled decreases in sialic acid. Aspirin treated patients and platelets incubated with aspirin in vitro both showed increased platelet EPM. These results suggest that platelet surface negative charge may directly affect platelet function.

The behavior of circulating 111In-labeled platelets in cerebrovascular injuries induced by arachidonic acid injection was studied. Fourteen rabbits were pretreated with the antiplatelet agent ticlopidine, and 10 rabbits were used for controls. Arachidonic acid (AA, 0.7 mg/kg) was injected into the internal carotid artery. Prior to the injection, platelets labeled with 111Inoxine were injected for autoradiography of the brain. Evans blue was injected as an indicator of blood-brain barrier disturbance. Nine control animals showed marked blue staining, and one showed slight blue staining. Seven out of the 14 pretreated animals showed slight or no staining, while 7 showed intensive staining. The distributions of the blue staining and the radioactivity showed high correlation. In the rabbits whose platelet aggregability was depressed by ticlopidine, lower blue staining as well as lower radioactivity was observed. Our findings suggest that activated platelets have an important role in the genesis of cerebrovascular injuries.

B16 mouse melanoma cell lines (B16F1, B16F10 and B16BL6) were able to induce platelet aggregation, and concomitant release of ATP in heparinized platelet-rich plasma (PRP). In citrated PRP, these tumor cells did not induce platelet aggregation. Addition of heparin to citrated PRP enabled these tumor cells to induce aggregation. In heparinized PRP, platelet aggregates induced by B16F10 cells were dissociated by the addition of either 4 mM EDTA, 10 mM CaCl2 or 0.1 micrograms/ml protamine sulfate. B16F10-induced aggregation in heparinized PRP was inhibited by preincubation with anti-fibronectin antibody, but not with antifibrinogen or anti-von Willebrand factor antibodies. B16F10 cells induced aggregation in washed platelet suspension with the addition of heparinized platelet-poor plasma (PPP). Cryoprecipitate from human plasma showed the same effect in the presence of heparin if substituted for PPP. The mixture of purified fibronectin, von Willebrand factor, fibrinogen and heparin were less effective than cryoprecipitate on B16F10-induced aggregation of washed platelets. The results suggest that an interaction between fibronectin and heparin may be important in tumor cell-induced aggregation.

Human platelets were separated into density subpopulations by using a step-wise gradient of Percoll in Tris-NaCl buffer. The absolute value of the electrophoretic mobility (EPM) of the density subpopulations was found to be a linear function of the density of the platelets, with EPM becoming less negative with increasing platelet density. Platelet volume distributions, mode volume, and sialic acid and protein contents were found to increase with platelet density, while no differences were found in GPII, GPIII, and GPIV contents among the subpopulations. An estimate of charge density was made from the ratio between the PAS-staining material (membrane GP's) and platelet surface area. The ratio was found to decrease as platelet density increased, consistent with the less negative EPM values observed for the higher density platelets. This lower surface charge of heavier platelets, which would lower charge repulsion between cells, agrees with the premise that heavier platelets are more active.