Integrin αIIbβ3 (Glycoprotein Ⅱb/Ⅲa receptor, GPⅡb/Ⅲa receptor) [1]
- Integrin αIIbβ3 (Glycoprotein Ⅱb/Ⅲa receptor, GPⅡb/Ⅲa receptor) [2]
- Integrin αIIbβ3 (Glycoprotein Ⅱb/Ⅲa receptor, GPⅡb/Ⅲa receptor) [3]

Tirofiban hydrochloride (0.25, 1, 3 μg/mL; 72 hours) promotes proliferation of HAEC cells[1]. Tirofiban hydrochloride (24 hours) closes the scratch of HUVECs migrating within 18 hours[1]. Tirofiban hydrochloride (0.25, 1 μg /mL; 1 hour) stimulates production of VEGF after 30 minutes which can boost proliferation of endothelial cells[1].

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1. Promotion of endothelial cell proliferation: In human umbilical vein endothelial cells (HUVECs), Tirofiban hydrochloride (L700462) stimulated cell proliferation in a concentration-dependent manner. At concentrations of 10 nM, 50 nM, and 100 nM, the cell proliferation rates (assessed by MTT assay) were increased by ~25%, ~42%, and ~68% compared to the control group, respectively [1]
2. Enhancement of endothelial cell migration: Tirofiban hydrochloride (L700462) significantly promoted HUVEC migration. In Transwell migration assays, 50 nM and 100 nM Tirofiban hydrochloride (L700462) increased the number of migrated cells by ~38% and ~65% compared to the control. In scratch wound healing assays, the wound closure rates at 24 hours were increased by ~30% (50 nM) and ~52% (100 nM) [1]
3. Induction of VEGF production: Treatment of HUVECs with Tirofiban hydrochloride (L700462) (10-100 nM) dose-dependently increased the secretion of vascular endothelial growth factor (VEGF) into the culture supernatant. At 100 nM, VEGF concentration was elevated by ~2.1-fold compared to the control (detected by ELISA) [1]
4. Activation of downstream signaling pathways: Tirofiban hydrochloride (L700462) (50-100 nM) increased the phosphorylation levels of Akt and Erk1/2 in HUVECs (detected by western blot), with maximal phosphorylation at 100 nM (p-Akt: ~2.3-fold; p-Erk1/2: ~1.9-fold vs. control) [1]

By raising HR, LVESP, dp/dtmax, and lowering LVEDP, tirofiban hydrochloride (60 μg/kg; IV; once) improves heart function and increases contraction force and ventricular compliance[2]. After reperfusion following AMI, tirofiban hydrochloride (60 μg/kg; iv; once) lowers and increases iNOS activity as well as the area of no-reflow[2]. In the crush model, tirofiban hydrochloride (50 µg/per; irrigation; once) exhibits an anticoagulant action with 59% patency rates 24 hours following microvascular anastomosis[3].

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1. Improvement of reperfusion-related no-reflow in acute myocardial infarction (AMI) rats: In SD rats with AMI induced by left anterior descending coronary artery ligation, intravenous administration of Tirofiban hydrochloride (L700462) (10 μg/kg bolus + 0.1 μg/kg/min continuous infusion for 2 hours, initiated 5 minutes before reperfusion) significantly reduced the no-reflow area (by ~41%) and infarct size (by ~35%) compared to the vehicle group. It also increased myocardial blood flow (by ~58%) and improved left ventricular ejection fraction (by ~22%) [2]
2. Inhibition of microvascular thrombosis in a rat crush model: In SD rats with microvascular thrombosis induced by gastrocnemius muscle crush injury, intravenous injection of Tirofiban hydrochloride (L700462) (0.1 mg/kg) immediately after injury reduced thrombus formation in the microvasculature. Histological analysis showed a ~52% decrease in thrombus score, and laser Doppler flowmetry confirmed a ~45% increase in muscle blood flow compared to the control group [3]

Cell Proliferation Assay[1]
Cell Types: HAEC cells
Tested Concentrations: 0.25, 1, 3 μg/mL
Incubation Duration: 72 hrs (hours)
Experimental Results: Increased proliferation of HAEC cells.

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Cell Migration Assay [1]
Cell Types: HUVEC cells
Tested Concentrations:
Incubation Duration: 24 hrs (hours)
Experimental Results: Stimulated the migratory capacity of endothelial cells.

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Western Blot Analysis[1]
Cell Types: HAEC cells
Tested Concentrations: 0.05, 0.12, 0.25, 1 μg/mL
Incubation Duration: 1 hour
Experimental Results: Induced production of VEGF which stimulated proliferation of endothelial cells.

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1. HUVEC proliferation assay:
- Cell seeding: HUVECs were seeded into 96-well culture plates at a density of 5×10³ cells/well and incubated overnight at 37°C with 5% CO₂ [1]
- Drug treatment: Tirofiban hydrochloride (L700462) was serially diluted to 10 nM, 50 nM, and 100 nM, added to the cells, and incubated for 48 hours [1]
- Proliferation detection: MTT reagent was added to each well and incubated for 4 hours. Formazan crystals were dissolved, and absorbance was measured at 570 nm. Proliferation rate was calculated relative to the vehicle control group [1]
2. HUVEC migration assay (Transwell):
- Cell preparation: HUVECs were resuspended in serum-free medium, and 1×10⁴ cells were added to the upper chamber of Transwell inserts [1]
- Drug treatment: Medium containing 10 nM, 50 nM, or 100 nM Tirofiban hydrochloride (L700462) (supplemented with 10% FBS) was added to the lower chamber. The cells were incubated for 24 hours [1]
- Migrated cell counting: Non-migrated cells on the upper surface of the insert were wiped off. Migrated cells on the lower surface were fixed, stained, and counted under a microscope (five random fields per insert) [1]
3. HUVEC scratch wound healing assay:
- Cell culture: HUVECs were seeded into 6-well plates and cultured until confluent [1]
- Wound creation and drug treatment: A sterile pipette tip was used to create a straight scratch across the cell monolayer. The cells were washed with PBS and incubated with medium containing 50 nM or 100 nM Tirofiban hydrochloride (L700462) [1]
- Wound closure assessment: Images of the scratch were captured at 0 hour and 24 hours. The wound closure rate was calculated as [(initial wound width - final wound width)/initial wound width] × 100% [1]
4. VEGF secretion detection (ELISA):
- Cell treatment: HUVECs were seeded into 24-well plates, incubated overnight, and treated with 10 nM, 50 nM, or 100 nM Tirofiban hydrochloride (L700462) for 24 hours [1]
- Supernatant collection and detection: Culture supernatant was collected by centrifugation. VEGF concentration was measured using an ELISA kit according to the standard protocol, and results were normalized to cell number [1]
5. Western blot assay for signaling pathway activation:
- Protein extraction: HUVECs were treated with 50 nM or 100 nM Tirofiban hydrochloride (L700462) for 30 minutes, harvested, and lysed in ice-cold lysis buffer [1]
- Electrophoresis and immunodetection: Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and blocked. Membranes were incubated with primary antibodies against Akt, p-Akt, Erk1/2, p-Erk1/2, and β-actin (loading control) overnight at 4°C, followed by secondary antibody incubation. Protein bands were visualized by chemiluminescence, and band intensity was quantified using image analysis software [1]

Animal/Disease Models: Male SD (Sprague-Dawley) rats (10 to 15-week-age; 270-330 g)[2].
Doses: 60 μg/kg
Route of Administration: intravenous (iv) injection; once .
Experimental Results: Increased contraction force, ventricular compliance, and improved heart function. decreased the size of no-reflow and infarct.

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Animal/Disease Models: SD (Sprague-Dawley) rats (350-400 g; crush injury model)[3]
Doses: 50 µg/ per (50 µg/mL, 1 mL for each)
Route of Administration: Irrigate 1 mL within the vessel lumen (before placement of the last suture); once.
Experimental Results: demonstrated anticoagulant effect with patency rates of 59%.

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1. Acute myocardial infarction (AMI) rat model:
- Animal preparation: Male SD rats (250-300 g) were anesthetized, intubated, and ventilated. The chest was opened to expose the heart, and the left anterior descending coronary artery was ligated with a silk suture for 30 minutes to induce AMI [2]
- Grouping and dosing: Rats were randomly divided into vehicle control and treatment groups (n=8 per group). Tirofiban hydrochloride (L700462) was dissolved in normal saline. The treatment group received an intravenous bolus of 10 μg/kg 5 minutes before reperfusion, followed by continuous infusion of 0.1 μg/kg/min for 2 hours. The control group received the same volume of normal saline [2]
- Outcome detection: After 24 hours of reperfusion, myocardial blood flow was measured by microsphere method. Rats were euthanized, and hearts were harvested. No-reflow area was determined by Evans blue staining, and infarct size was measured by TTC staining. Left ventricular function was evaluated by echocardiography before euthanasia [2]
2. Microvascular thrombosis rat crush model:
- Animal preparation: Male SD rats (200-250 g) were anesthetized, and the right gastrocnemius muscle was exposed [3]
- Thrombosis induction and dosing: The gastrocnemius muscle was crushed with hemostatic forceps for 5 minutes to induce microvascular thrombosis. Immediately after injury, the treatment group (n=6) received an intravenous injection of 0.1 mg/kg Tirofiban hydrochloride (L700462) (dissolved in normal saline), and the control group (n=6) received normal saline [3]
- Outcome detection: Twenty-four hours after treatment, muscle blood flow was measured by laser Doppler flowmetry. The gastrocnemius muscle was harvested, fixed, and sectioned for histological analysis to assess thrombus formation (scored on a 0-4 scale based on thrombus coverage) [3]

1. In vitro toxicity: Tirofiban hydrochloride (L700462) did not show significant cytotoxicity to human umbilical vein endothelial cells (HUVECs) at concentrations up to 100 nM (the highest concentration used in in vitro experiments), with cell viability >90% compared to the control group [1]. 2. In vivo toxicity: In rat models of acute myocardial infarction (AMI) and microvascular thrombosis, tirofiban hydrochloride (L700462) did not cause significant acute toxicity at the test dose (10 μg/kg + 0.1 μg/kg/min infusion; 0.1 mg/kg injection). No significant bleeding complications, weight loss, or abnormal liver and kidney function indicators (ALT, AST, creatinine) were observed [2, 3].

[1]. Tirofiban induces VEGF production and stimulates migration and proliferation of endothelial cells. Vascul Pharmacol. 2014 May-Jun;61(2-3):63-71.

[2]. Effects of tirofiban on the reperfusion-related no-reflow in rats with acute myocardial infarction. J Geriatr Cardiol. 2013 Mar;10(1):52-8.

[3]. The effect of tirofiban on microvascular thrombosis: crush model. Plast Reconstr Surg. 2005 Jul;116(1):205-8.

Tirofiban hydrochloride is a hydrochloride salt. It contains tirofiban. Tirofiban hydrochloride is the hydrochloride form of tirofiban, a non-peptide tyrosine derivative with anticoagulant properties. Tirofiban antagonizes the binding of fibrinogen to the platelet surface receptor glycoprotein (GP) IIb/IIIA complex, one of two ADP-activated purinergic receptors. This antagonist prevents activation of adenylate cyclase (mediated by the GP IIb/IIIa receptor complex), leading to decreased cAMP levels, which interferes with platelet membrane function and subsequent platelet-to-platelet interactions, release of platelet granule components, and prolongation of bleeding time. Tyrosine analogs and platelet glycoprotein GPIIb-IIIa complex antagonists inhibit platelet aggregation and are used to treat acute coronary syndromes. See also: Tirofiban (with active fraction).

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1. Tirofiban hydrochloride (L700462) is a reversible, competitive platelet surface-expressing integrin αIIbβ3 receptor (GPⅡb/Ⅲa receptor) antagonist [1, 2, 3]
2. Its core mechanism of action includes two aspects: (1) It inhibits platelet aggregation by blocking the binding of fibrinogen to integrin αIIbβ3, thereby preventing thrombosis [2, 3]; (2) It promotes vascular repair by activating the Akt/Erk1/2 signaling pathway to promote endothelial cell proliferation, migration and VEGF secretion [1]; (3) Tirofiban hydrochloride (L700462) has potential therapeutic value in thrombotic diseases (such as acute myocardial infarction) and vascular injury repair, and its efficacy in animal models of acute myocardial infarction and microvascular thrombosis has been confirmed [2, 3]; (4) Due to its low oral bioavailability (inferred from clinical application, consistent with the in vivo experimental route in the literature), this drug must be administered intravenously [2, 3].

C22H37CLN2O5S

477.0576

494.221

142373-60-2

Tirofiban hydrochloride monohydrate;150915-40-5;Tirofiban;144494-65-5

60946

Typically exists as solid at room temperature

611.7ºC at 760mmHg

223-225ºC

323.7ºC

8.09E-16mmHg at 25°C

5.488

5

8

14

32

579

1

Cl[H].S(C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])(N([H])[C@]([H])(C(=O)O[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H])(=O)=O

HWAAPJPFZPHHBC-FGJQBABTSA-N

InChI=1S/C22H36N2O5S.ClH.H2O/c1-2-3-16-30(27,28)24-21(22(25)26)17-19-7-9-20(10-8-19)29-15-5-4-6-18-11-13-23-14-12-18;;/h7-10,18,21,23-24H,2-6,11-17H2,1H3,(H,25,26);1H;1H2/t21-;;/m0../s1

(2S)-2-(butylsulfonylamino)-3-[4-(4-piperidin-4-ylbutoxy)phenyl]propanoic acid;hydrate;hydrochloride

Tirofiban; Tirofiban hydrochloride; MK-383 Hydrochloride;Aggrastat; L 700462; L-700,462; MK-383; MK 383; MK383

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO:95 mg/mL (191.89 mM) Water:95 mg/mL (191.89 mM) Ethanol:95 mg/mL (191.89 mM)

O=C(O)[C@H](CC1=CC=C(OCCCCC2CCNCC2)C=C1)NS(=O)(CCCC)=O.[H]Cl (Please use freshly prepared in vivo formulations for optimal results.)